Controlling attention and memory by altering neuronal carbonic anhydrase activity

ABSTRACT

The invention provides a method for modulating attentive cognition comprising administering a compound that alters intraneuronal carbonic anhydrase activity thereby affecting establishment of a theta rhythm. The metabolic pathway of the compound preferably involves bicarbonate-mediated GABAergic depolarization. The term “attentive cognition” is meant to encompass memory formation, learning, spatial memory, and attention. The modulating may be stimulating, or the compound may have the multiple effects of inhibiting intraneuronal carbonic anhydrase activity, establishment of a theta rhythm, and memory acquisition. The invention further provides a method of modulating memory and attention comprising switching theta rhythm on and off, the switching comprising potentiating or inhibiting intraneuronal carbonic anhydrase activity.

This application claims the benefit of provisional application USSN60/209,790, filed Jun. 7, 2000, incorporated herein by reference.

BACKGROUND OF THE INVENTION

This invention relates to methods for modulating attention, learning,and memory by controlling carbonic anhydrase activity. Moreparticularly, the invention involves administering a compound thatalters carbonic anhydrase activity in the brain thereby affectingestablishment of a theta rhythm in the brain.

Acetazolamide is a generic drug manufactured in forms of capsule,tablets or injectable preparations. Inhibition of carbonic anhydraseactivity by acetazolamide is a therapeutic approach in use or suggestedfor several indications, including glaucoma, conjunctive heart failure,mountain sickness, sleep apnea and petit mal seizures (epilepsy), andbody fluid retention. These therapeutic avenues are diverse and none ofthem suggests any cognitive impacts of regulating carbonic anhydraseactivity.

Hippocampal theta (θ) rhythm (synchronized neuronal discharge) isbelieved to play a critical role in information processing and memoryconsolidation during exploratory behavior. Theta activity depends oncholinergic inputs and θ discharges of GABAergic interneurons, and canbe induced e.g. by a cholinergic receptor agonist (carbachol). GABA actsas an inhibitor of action potentials by keeping the GABA-Cl⁻ channelopen. This channel is an anion transporter, exchanging Cl⁻ for HCO₃ ⁻.Maintaining the open channel allows Cl⁻ to flow into the cell tohyperpolarize the membrane potential. Cholinergic components depolarizethe membrane potential, and if threshold is reached an action potentialis created. It is believed that the synchronized depolarization andexcitation creates an oscillating membrane potential leading to θactivity, but the mechanism has remained unknown. GABAergicdepolarization can be induced by enhancing HCO₃ ⁻ conductance throughGABA_(A) receptor-channels in adult hippocampal cells, a responsesensitive to carbonic anhydrase inhibitors (Kaila '93). However, noassociation has previously been identified between carbonic anhydraseinhibition at the cellular-electrophysiological level on the one hand,and a reduction in theta rhythm on the other.

There have been few medications suitable for improving attention inthose who need it, and no effective medications for those who need tosuppress learning painful memories. There is a need to elucidate asimple biochemical target for controlling the extremely complicatedbrain-wide effect of theta rhythm, and the even more complex eventsleading to attention and learning. Such a target would be invaluable inidentifying specific compounds for achieving the desired cognitiveeffects.

This invention differs from the prior art in modifications, which werenot previously known or suggested, including the use of carbonicanhydrase regulators to alter theta rhythm and produce cognitive effectsin mammals. The methods of the invention provide advantages that werenot previously appreciated, such as the ability to selectively enhanceattention and learning. This invention satisfies a long felt need forcompounds that selectively enhance or inhibit attention and learning.

The widespread use of acetazolamide, with no substantial cognitive sideeffects reported, makes it surprising to discover that the compound doesinhibit spatial learning in animals, and presumably in humans. Thisinvention thus identifies a previously unrecognized problem, and showshow to solve it, e.g. by careful dosing regimens of acetazolamide so asto reduce its cognitive side effects.

SUMMARY OF THE INVENTION

Acetazolamide, a drug commonly used for glaucoma and diuresis, has beenfound to specifically block acquisition of new memories. Post-traumaticstress disorder involves learning new contexts for traumatic memories.These new contexts would be blocked during the administration ofacetazolamide. Thus, acetazolamide can be used for short-term memorysuppression, for example to prevent post traumatic stress syndromeduring a period of trauma.

More generally, based on comprehensive electrophysiologic studies ofGABAergic depolarization of CA1 hippocampal cells, we have implicatedbicarbonate conductance enhancement as critical for theta rhythm andspatial maze learning. Bicarbonate-mediated GABAergic depolarizationmediated by carbonic anhydrase (blocked by acetazolamide) is a targetfor drugs that either block or enhance new memory formation. Blockingdrugs would have application in post-traumatic stress disorders andrelated diseases. Cognitive enhancing drugs would have wide applicationin the treatment of neurodegenerative disorders, especially thoseinvolving dementia.

Surprisingly, according to the invention, the GABAergic transformationis associated with establishment of a theta rhythm, and both phenomenacan be controlled together by modifying neuronal carbonic anhydraseactivity. Although it was previously thought that the transformation wasrelated to attention, and that the theta rhythm was associated withattention, there was no teaching that the two were intimately connectedand subject to common control via carbonic anhydrase inhibitors oractivators.

The invention relates to a method for blocking associative memoryacquisition comprising determining a need for blocking, andadministering an inhibitor, such as acetazolamide, of carbonic anhydraseactivity in the brain, thereby blocking the memory acquisition. Themethod can also be used for suppressing attention, learning and/ormemory formation in a mammal comprising determining a need forsuppressing, and administering an inhibitor of carbonic anhydraseactivity in an amount effective to inhibit carbonic anhydrase activityin the brain, preferably in neurons. The inhibitor may be selected fromthe group consisting of acetazolamide, benzolamide, and analogs thereof.An analog is a molecule having a structure function relationship similarto the named compound that allows it to bind to and inhibit carbonicanhydrase in the brain. Although, the inhibitor prevents establishmentof a theta rhythm during learning, it does not affect memory retrievalfrom formed memories, or sensory or locomotor behaviors.

In yet another aspect of the invention, the method can be used forimproving attention and/or memory acquisition in a patient comprisingdetermining the need for improved attention and/or memory acquisition,and administering to the patient a stimulator of intraneuronal carbonicanhydrase activity in an amount sufficient to stimulate intraneuronalcarbonic anhydrase activity. The patient may be healthy, i.e. have noneurodegenerative disorder or the patient may suffer from aneurodegenerative disease. The invention may also be used for treating aneurodegenerative disorders comprising administering an effective amountof a stimulator of intraneuronal carbonic anhydrase activity. Thecognitive ability may be enhanced. The neurodegenerative disease canalso be dementia.

The invention further provides a method for modulating attentivecognition comprising administering a compound that alters intraneuronalcarbonic anhydrase activity in an amount sufficient to affect theestablishment of a theta rhythm. The theta rhythm can be affected bymodulating bicarbonate-mediated GABAergic depolarization. Moreover, theattentive cognition may be selected from memory formation, learning,spatial memory, and attention. In one aspect the modulating isstimulating. The compound can be administered in an amount sufficient toinhibit intraneuronal carbonic anhydrase activity, establish of a thetarhythm, and suppress memory acquisition. The compound may also beadministered in an amount that does not affect memory retrieval.Moreover, the suppression may occur about one half to one hour afteradministering the inhibitor compound.

The term “attentive cognition” is meant to encompass memory formation,learning, spatial memory, and attention. The inhibiting of carbonicanhydrase is preferably selective in that it reduces memory acquisition,but not memory retrieval.

In another aspect of the invention, the method may be used formodulating memory and attention by switching theta rhythm on and off tomodulate intraneuronal carbonic anhydrase activity, comprisingadministering a compound that modulates intraneuronal carbonic anhydraseactivity. The modulating intraneuronal carbonic anhydrase activity maycomprise increasing intraneuronal carbonic anhydrase activity oralternatively decreasing intraneuronal carbonic anhydrase activity.

In yet a further aspect, the invention relates to a method of alteringmemory acquisition by modulating HCO₃ ⁻ conductance comprisingadministering a compound that modulates carbonic anhydrase activity thebrain in an amount sufficient to alter HCO₃ ⁻ conductance. The compoundcan be administered in an amount sufficient to modulate the HCO₃ ⁻current relative to the Cl⁻ and K⁺ currents. The invention also relatesto a method of modulating establishment of a theta rhythm comprisingadministering a compound that modulates intraneuronal carbonic anhydraseactivity in an amount sufficient to control the occurrence of synaptictransformation. Another aspect of the invention relates to a method fortreating a neurological disorder comprising administering a compoundthat stimulates intraneuronal carbonic anhydrase activity in an amountsufficient to control the occurrence of synaptic transformation. Theneurological disorder can be associated with a disorder affectingcognition such as stroke, hypoxia, and ischemia, and others known topractitioners.

The invention may also relate to a method for screening compounds forusefulness for cognitive enhancement therapy comprising measuring theeffect of a compound on carbonic anhydrase activity in neurons, intissue, in animals, or in cell culture, and selecting those compoundsthat stimulate carbonic anhydrase activity. In addition, the inventionprovides a method for screening compounds for usefulness for cognitiveenhancement therapy comprising measuring the effect of a compound ontheta rhythm in neurons, in tissue, in animals, or in cell culture andselecting those that establish a theta rhythm.

Finally, the invention provides a method of blocking synaptictransformation of inhibitory postsynaptic potentials into excitatorypostsynaptic potentials in GABAergic synapses in a mammalian brain,comprising determining a need for blocking, and administering to thebrain an inhibitor of intraneuronal carbonic anhydrase activity, therebyblocking the synaptic transformation in the synapses. The inhibitor mayneutralize the excitatory effects of a memory related signaling protein.The memory related signaling protein can be calexcitin and the inhibitorcan be acetazolamide or an analog of acetazolamide. The metabolicpathway of the compound preferably involves bicarbonate-mediatedGABAergic depolarization. The affected synapses may be found in circuitsthroughout the brain, including pyramidal cells in the hippocampalregion.

The invention also relates to a pharmaceutical product comprising adosage form comprising a pharmaceutically acceptable carrier and acompound that inhibits or stimulates carbonic anhydrase activity in thebrain, associated with labeling indicating use of the dosage form forcognitive effects.

Further objectives and advantages will become apparent from aconsideration of the description, drawings, and examples.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is better understood by reading the following detaileddescription with reference to the accompanying figures, in which likereferences refer to like elements throughout, and in which:

FIGS. 1A1 to 1A3, 1B1 to 1B4 and 1C1 to 1C4 show the effects ofcarbachol (CCH)-induced θ oscillations of hippocampal CA1 fieldpotential and of membrane potential of CA1 pyramidal cells, which dependon activation of GABAergic inputs.

FIGS. 2A1 to 2A5, 2B and 2C demonstrate the effects of carbachol(CCH)-induced θ oscillations of hippocampal CA1 field potential and ofmembrane potential of CA1 pyramidal cells, which are associated withGABAergic postsynaptic depolarization.

FIGS. 3A1 to 3A3, 3B1 to 3B3 and 3C1 to 3C3 show carbachol shiftsreversal potentials of GABAergic postsynaptic responses in hippocampalCA1 pyramidal cells.

FIGS. 4A1 to 4A3, and 4B1 to 4B3 demonstrate the effects of carbachol(CCH)-induced θ oscillations of hippocampal CA1 field potential and ofmembrane potential of CA1 pyramidal cells, which depend on HCO₃ ⁻formation.

FIGS. 5A, 5B and 5C1 to 5C2 show the effects of intracellularadministration of calexcitin associated with postsynaptic depolarizationinduced intracellular θ in hippocampal pyramidal cells.

FIGS. 6A, 6B, 6C and 6D demonstrate rebound action potentials ofhippocampal CA1 pyramidal cells evoked by GABAergic inhibition, whichvary in occurrence and timing.

FIGS. 7A1 to 7A3, 7B1 to 7B2 and 7C1 to 7C5 demonstrate the effects ofcarbachol (CCH)-induced θ GABAergic depolarization of hippocampal CA1pyramidal cells in enabling GABAergic inputs to entrain CA1 pyramidalcells.

FIGS. 8A, 8B, 8C1 to 8C2, 8D1 to 8D2 and 8E show the effects of carbonicanhydrase inhibitors in impairing rat formation of spatial memory invivo.

FIGS. 9A, 9B1 to 9B2 and 9C1 to 9C2 demonstrate the effects ofacetazolamide administration in not affecting retrieval of formedspatial memory.

FIGS. 10A to 10H, 10I1 and 10I2, and 10J to 10O show the effects of CEin transforming BAS-CA1 synapses.

FIGS. 11A to 11E demonstrate the effects of anti-CE antibody inenhancing BAS-CA1 IPSPs, and provide potential mechanisms of CE-inducedtransformation of GABAergic synapses.

FIGS. 12A to 12K demonstrate the effects of ACET and non-bicarbonatebuffer in eliminating CE-induced transformation, which convertsexcitatory input filter into amplifier.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In describing preferred embodiments of the present invention illustratedin the drawings, specific terminology is employed for the sake ofclarity. However, the invention is not intended to be limited to thespecific terminology so selected. It is to be understood that eachspecific element includes all technical equivalents, which operate in asimilar manner to accomplish a similar purpose. Each reference citedhere is incorporated by reference as if each were individuallyincorporated by reference.

Attentive cognition according to the invention means the ability to payattention, to learn, and/or to form and acquire memory, includingencoding experience into memory and forming associative memory, such asspatial memory, or memories of auditory and/or visual images. Preferablythe attentive cognition effects according to the invention are specific.Thus, suppressing attentive cognition preferably does not suppressmemory retrieval from stored memories, or cause sensory or locomotoreffects.

The invention provides therapeutic methods for modulating attentivecognition by modulating carbonic anhydrase activity in the brain. Inparticular, the core pathway employed by the inventive methods may besummarized as follows:

Carbonic Anhydrase→Attentive Cognition

More particularly, preferred aspects of the core pathway which aremodulated by the methods of the invention are as follows:

Carbonic Anhydrase→bicarbonate formation→Synaptic transformation→Thetarhythm→Attentive Cognition→behavior effects or disease therapy

The inventive methods and compositions intervene at various stages inthe aspects of the core pathway, and indeed the invention contemplateseach of the various combinations of the aspects of the pathway. Forexample, the invention contemplates methods of increasing or decreasingnot only carbonic anhydrase activity, but also (or instead) increasingor decreasing bicarbonate formation, synaptic transformation, and/ortheta rhythm establishment to provide some or all of the listeddownstream effects, including increasing or decreasing bicarbonateformation, synaptic transformation, and/or theta rhythm establishment,and the ultimate effects including the presence of attentive cognition,and resulting behavioral effects and therapies for disease.

By increasing GABA_(A) receptor-channel HCO₃ ⁻ current relative to Cl⁻current, GABA induced depolarization is promoted. This mechanism issensitive to carbonic anhydrase inhibitors. θ activity is dependent onGABAergic postsynaptic depolarization and a shift of the reversalpotential from Cl⁻ towards HCO₃ ⁻. The cholinergic θ activity involves aswitch of GABAergic postsynaptic responses from hyperpolarizingpredominantly Cl⁻ to a depolarizing HCO₃ ⁻ conductance. This reversedpolarity can effectively and immediately entrain pyramidal cells into aθ rhythm.

In vitro, the θ activity was shown to be abolished by GABA_(A) receptorantagonists and carbonic anhydrase inhibitors, but largely unaffected byblocking cholinergic receptors. Acetazolamide was used to inhibitcarbonic anhydrase, an enzyme that increases HCO₃ ⁻ formation andregulates HCO₃ ⁻ ionic gradient. Carbonic anhydrase is present inhippocampal pyramidal cells. In vivo, carbonic anhydrase inhibition alsoimpaired spatial learning in a watermaze, but did not affect othersensor/locomotor behaviors.

Thus, HCO₃ ⁻-mediated signaling, as regulated by carbonic anhydrasethrough reversed polarity of GABAergic postsynaptic responses, isimplicated in both θ activity and memory consolidation in rat spatialmaze learning. Carbonic anhydrase activity appears to be an essentialrequirement in the molecular signaling pathways of GABAergic synapses.The multiple and related effects of carbonic anhydrase in cognition, andthe ability to control them via inhibition of the enzyme, are surprisingresults, and of great significance in designing therapeutic targets.

According to the invention, experiments show that reversed HCO₃⁻-dependent GABAergic postsynaptic responses and their effectiveness inentraining θ activity of pyramidal cells play a central role in memoryretention. Carbonic anhydrase is very efficient and may act as afunctional switch to turn on and off θ activity, thereby controllingmemory retention. acetazolamide-regulated HCO₃ ⁻ gradients appearimportant for acquisition of memory rather than retrieval from formedmemory. Such compounds have clinical value when temporarily suppressedmemory is beneficial (e.g. surgery or post-traumatic-stress-disorder).

An aspect of the invention relates to a method of treatment usingacetazolamide as a memory suppressor to treat posttraumatic syndrome.The dosage used in the memory experiments provides concentrations of1-10 micromolar, and in rats corresponds to about 15 mg/Kg. Thiscorresponds to about 1 g for a 70 kg human for a single dosage.Acetazolamide is currently available in dosage forms including 500 mgcapsules and injectables; and 250-mg capsules or tablets. Thus, theeffectiveness of the inventive method is readily predictable from theanimal studies. Moreover, rat spatial learning tests are known to bepredictive of attention and learning in humans. Furthermore, it is knownthat theta rhythms in humans are associated with learning. Thus, thedata presented in the examples establish a strong basis forextrapolating to effective therapies for humans.

Example 1 (Miao-Kun Sun, Wei-Qin Zhao, Thomas J. Nelson, and Daniel L.Alkon, “Theta Rhythm of Hippocampal CA1 Neuron Activity: Gating byGABAergic Synaptic Depolarization,” Journal of Neurophysiology, vol. 85No. 1, pp. 269-279 (January 2001)), which is incorporated herein byreference in its entirety, establishes that carbonic anhydrase increasesHCO₃ ⁻ formation and regulates HCO₃ ⁻ gradient formation, to produceGABAergic postsynaptic response and establish a theta rhythm.Memory-related signaling, encoding an experience into lasting memory,and associative memory are linked to the establishment of a thetarhythum. It is thought that an increase in HCO₃ ⁻ is needed to mediateattention. These effects seem to control memory acquisition andattention. Acetazolamide inhibits carbonic anhydrase, and reduces HCO₃ ⁻levels. It has newly been discovered that acetazolamide blocks the thetarhythm, and thereby prevents memory acquisition. Intracellularbenzolamide, which does not cross the cell membrane absent injection,causes a similar effect when injected. The structural homology of thetwo compounds suggests a family of analogs with similar effects, aswould be known to a person of ordinary skill.

As detailed below, Example 2 relates to the synaptic transformation ofGABAergic interneuron cells from producing inhibitory post synapticsignals (IPSPs) to producing excitatory post synaptic signals (EPSPs).It has been determined that calexcitin reduces IPSPs, and has effects onK⁺-channels, does not blockade a relevant receptor-channel complex, anddoes cause shifts of reversal potential. Confirming the excitatoryeffect of calexcitin, anti-CE antibodies enhance IPSPs. Moreparticularly, it has been learned that acetazolamide eliminatescalexcitin-induced alterations in IPSPs, probably due to the carbonicanhydrase type II isoform. Acetazolamide at 10 micromolar, for 30minutes, is sufficient to maintain IPSPs while eliminatingtransformative effects of calexcitin. This teaches a central role ofHCO₃ ⁻/carbonic anhydrase activity in the calexcitin-induced synaptictransformation. Calexcitin had no direct effect on carbonic anhydraseactivity in a homogenate and so does not act directly on the enzyme.

The effects of carbonic anhydrase activity on inhibiting and/orstimulating theta rhythm and memory formation were not previously knownor suggested and are of great significance. For example, as a person ofordinary skill can understand, any carbonic anhydrase inhibitor,including suitable structural analogs and derivatives of acetazolamideand benzolamide, would have a similar memory suppressing effect. In thepresence of acetazolamide, Inhibitory Post-Synaptic Potentials (IPSPs)are not converted well to Excitatory Post-Synaptic Potentials (EPSPs).Likewise, potentiators, or upregulators of carbonic anhydrase increasethe establishment of a HCO₃ ⁻ gradient and theta rhythm, and produce astate of enhanced attention and learning.

According to the invention, one can administer a drug to a patient at agiven time to produce a cognitive effect (referred to as attentivecognition), such as learning, learning-related attention, associativelearning, and memory acquisition, and memory consolidation (withoutaffecting memory storage and recall) by modulating neuronal carbonicanhydrase activity. The modulation may be inhibition, e.g byacetazolamide and other carbonic anhydrase inhibitors, or it may beexcitation, e.g. by compounds that enhance carbonic anhydrase activityand thereby switch GABAergic activity from predominantly hyperpolarizingCl⁻ conductance to a depolarizing, primarily HCO₃ ⁻ conductance,entraining pyramidal cells into a theta rhythm.

The compounds administered according to the invention can inhibitGABAergic depolarization, and reduction of the theta rhythm, and in turndiminish memory and attention (or the opposite). Without intending tolimit the scope of the invention, it is believed that the followingmechanism is at play according to the invention. GABA opens Cl⁻ and K⁺channels. Bicarbonate ion HCO₃ ⁻ flows through the same channels. Whenthere is relatively more bicarbonate in the cell, it flows out, changingthe charge-carrying capacity to bicarbonate from chloride ion.Inhibiting carbonic anhydrase reduces bicarbonate availability relativeto chloride ion, thus causing a relative hyperpolarizing effect, anddiminishing GABAergic response and synaptic transformation. Activatingcarbonic anhydrase increases bicarbonate concentration, and/or itsavailability to flow out through the Cl⁻ and/or K⁺ channels, thus havinga depolarizing effect, increasing synaptic transformation in the brainand in turn, theta rhythm and attentive cognition.

According to the invention, activators of carbonic anhydrase that arepresently known or subsequently discovered may be administered to apatient to increase neuronal carbonic anhydrase activity and to producethe resulting desired effects. This method follows from the method ofadministering a carbonic anhydrase inhibitor as demonstrated in theexamples.

Neuronal carbonic anhydrase activity is meant to encompass activity ofcarbonic anhydrase in the brain, preferably intraneuronally.Alternatively the invention contemplates modulating extraneuronalcarbonic anhydrase. The locus of action may be in various regions of thebrain, preferably and as demonstrated in the hippocampus.

The established electrophysiological and biochemical effects of carbonicanhydrase inhibitors on the synaptic transformation of GABAergicsynapses support the basic method of inhibiting synaptic transformationin hippocampal cells by administering a carbonic anhydrase inhibitor.The inhibitor eliminates the stimulative effect of calexcitin on cellsin hippocampal tissue slices. Further, acetazolamide counteracts thestimulative effects of carbachol, a cholinergic receptor agonist, invitro.

As to the theta rhythm gating described in more detail in the examples,it is known that theta rhythm is associated with attention, one aspectof associative learning. However, the theta rhythm effects of carbonicanhydrase inhibitors demonstrated here are new and different from whatwas previously known. Prior to this invention, there was no motivationto combine the electrophysiology and cellular biochemistry relating tocarbonic anhydrase with studies of theta rhythm, as they relate to twodistinct fields of study.

Thus, principal aspects of the invention include (1) specific cognitiveeffects, (2) theta rhythm effects, and in particular, (3) the method ofenhancing learning by stimulating carbonic anhydrase activity, and (4)inhibiting attention below standard control levels. The fact thatcarbonic anhydrase is a common link between stimulating excitatory postsynaptic potential and stimulating theta rhythm is completelyunprecedented and extremely useful in allowing therapies forneurological disorders, including cognitive therapy.

While the studies relate to hippocampal CA1 pyramidal cells, theinvention applies to any susceptible target cells in the brain, i.e.GABAergic synaptic circuits.

Interestingly, widespread use of acetazolamide e.g. for glaucoma may becausing memory blockage as an unidentified side effect. The new resultssuggest screening for those side effects and perhaps counteracting themwith cognitive enhancers, or precise dosage and timing regimes thatminimize the effects.

EXAMPLES Example 1

Information processing and memory consolidation during exploratorybehavior require synchronized activity known as hippocampal theta (θ)rhythm. Theta (θ) activity depends on cholinergic inputs from the medialseptum/vertical limb of the diagonal band nucleus (MS/DBv) and θdischarges of GABAergic interneurons, and can be induced withcholinergic receptor agonists. However, it was not clear how theincreased excitation of pyramidal cells could occur with increaseddischarges of GABAergic interneurons during θ waves. The followingexperiments show that the characteristic θ activity in adult rathippocampal CA1 pyramidal cells is associated with GABAergicpostsynaptic depolarization and a shift of the reversal potential fromCl⁻ toward HCO₃ ⁻ (whose ionic gradient is regulated by carbonicanhydrase). The θ activity was abolished by GABA_(A) receptorantagonists and carbonic anhydrase inhibitors, but largely unaffected byblocking glutamate receptors. Carbonic anhydrase inhibition alsoimpaired spatial learning in a watermaze without affecting othersensory/locomotor behaviors. Thus, HCO₃ ⁻-mediated signaling, asregulated by carbonic anhydrase, through reversed polarity of GABAergicpostsynaptic responses is implicated in both θ and memory consolidationin rat spatial maze learning. These experiments suggest that thismechanism may be important for the phase forward shift of the place celldischarges for each θ cycle during the animal's traversal of the placefield for that cell.

Introduction

Synchronization of neural activity within mammalian brain structures, asoccurs during hippocampal θ rhythm (Skaggs W E, and McNaughton B L.,Replay of neuronal firing sequences in rat hippocampus during sleepfollowing spatial memory., Science 271: 1870-1873, 1996; Huerta, P. T.and Lisman, J. E., Heightened synaptic plasticity of hippocampal CA1neurons during a cholinergically induced rhythmic state, Nature 364:723-725, 1993; O'Keefe, J. and Recce, M. L., Phase relationship betweenhippocampal place units and the EEG theta rhythm, Hippocampus 3:317-330, 1993; Shen, J., Barnes, C. A., McNaughton, B. L. Skaggs, W. E.,and Weaver, K. L., The effects of aging on experience-dependentplasticity of hippocampal place cells, J. Neurosci. 17: 6769-6782,1997), contributes to diverse forms of information coding (Draguhn, A.,Traub, R. D., Schmitz, D., and Jefferys, J. G., Electrical couplingunderlies high-frequency oscillations in the hippocampus in vitro,Nature 394: 189-192, 1998; Usher, M. and Donnelly, N., Visual synchronyaffects binding and segmentation in perception, Nature 394: 179-182,1998; Rodriguez, E., George, N., Lachaux, J. P., Martinerie, J.,Renault, B., and Varela, F. J., Perception's shadow: long-distancesynchronization of human brain activity, Nature 397: 430-433, 1999). Theθ frequency field oscillation, a major feature of the hippocampalelectroencephalogram (EEG), for example, occurs during two specificbehaviors, exploration and rapid-eye-movement (REM) sleep, and reflectssynchronized synaptic potentials that entrain the discharge of neuronsat frequencies between 4 and 12 Hz. The rhythm is believed by many togate or facilitate memory information processing in the hippocampus,particularly during persistent information storage. Thus, as an animalexplores its environment, MS/DBv cholinergic inputs, which innervate thewhole hippocampal formation (Dutar, P., Bassant, M. -H., Senut, M. -C.,and Lamour, Y, The septohippocampal pathway: structure and function of acentral cholinergic system, Physiol. Rev. 75: 393-427, 1995; Vertes, R.P. and Kocsis, B., Brainstem-diencephalo-septo-hippocampal systemscontrolling the theta rhythm of the hippocampus, Neuroscience 81:893-926, 1997), activate hippocampal θ rhythm (Vertes and Kocsis 1997).Briefly increased θ power has been reported during a word recognitionmemory task in humans, with a delay of about 125 ms after the visualpresentation of a word (Burgess, A. P. and Gruzelier, J. H., Shortduration synchronization of human theta rhythm during recognitionmemory, NeuroReport 8: 1039-1042, 1997). Recording neuromagnetic signalsduring a working memory task in humans reveals stimulus-lockedhippocampal θ (Tesche, C. D. and Karhu J., Theta Oscillations IndexHuman Hippocampal Activation During a Working Memory Task, Proc. Natl.Acad. Sci. USA 97:919-924, 2000). Evidence has also been provided thatdisruption of the θ activity by lesions of cholinergic inputs to thehippocampus blocks spatial memory (Winson, J., Loss of hippocampal thetarhythm results in spatial memory deficit in the rat, Science 201:160-163, 1978). The synaptic bases of the θ rhythm have been extensivelystudied, but many important questions related to the underlyingmechanism(s) for the θ activity remain to be answered. For instance,while the cholinergic θ activity recorded in place pyramidal cells isknown to depend on θ rhythmic activity from GABAergic interneurons,pyramidal cells are excited when the animals travel into the field ofthe place cell, i.e. when GABAergic interneurons are most active(Soltesz, I. and Deschenes, M., Low- and high-frequency membranepotential oscillations during theta activity in CA1 and CA3 pyramidalneurons of the rat hippocampus under ketamine-xylazine anesthesia, J.Neurophysiol. 70: 97-116, 1993; Ylinen, A., Soltesz, I., Bragin, A.,Penttonen, M., Sik, A., and Buzsaki, G., Intracellular correlates ofhippocampal theta rhythm in identified pyramidal cells, granule cells,and basket cells, Hippocampus 5: 78-90, 1995; Cscsvari, J., Hirase, H.,Czurkd, A., Mamiya, A., and Buzsaki, G., Oscillatory coupling ofpyramidal cells and interneurons in the behaving rat, J. Neurosci. 19:274-287, 1999). Furthermore, the firing period of the place cell duringthe exploration traversal shifts forward during each θ wave and becomesmore in phase with interneuron discharge.

Here, it is shown that cholinergic θ activity in hippocampal CA1pyramidal cells involves a switch of GABAergic postsynaptic responsesfrom a predominantly hyperpolarizing Cl⁻ to a depolarizing,predominantly HCO₃ ⁻ conductance. GABAergic activity through thereversed polarity can effectively and immediately entrain the pyramidalcells into a θ rhythm. Reducing HCO₃ ⁻ formation by inhibition ofcarbonic anhydrase blocks θ rhythm induction in vitro and impairs ratwatermaze performance in vivo. Switching between these operationalstates of the synapses may thereby provide a powerful way to selectivelydirect signal processing through the network.

Methods

Chemicals. Agents were either injected into the recorded cells throughthe recording electrodes: benzolamide (gift from T. H. Maren, Universityof Florida, Gainesville; 0.1 mM; 0.5 nA, 500 ms at 50% on cycles for 10min) and calexcitin (260 ng/μl of cloned calexcitin in 1 M K acetate, pH7.4; −2.0 nA, 700 ms at 33% on cycles for 15 min), or through theperfusion medium: kynurenic acid (Sigma), bicuculline methiodide (BIC;Sigma); carbachol (CCH; Sigma), acetazolamide (ACET; Sigma), andatropine sulfate (Sigma).

Hippocampal slice electrophysiology. CA1 field potentials were recordedwith glass microelectrodes filled with an artificial cerebrospinal fluidsolution (ACSF; see below). Male Sprague-Dawley rats (150-200 mg) weredecapitated, and the brains were removed and cooled rapidly in an ACSFsolution (˜4° C.), bubbled continuously with 95% O₂-5% CO₂. Hippocampiwere sliced (400 μM), placed in oxygenated ACSF (in mM: 124 NaCl, 3 KCl;1.3 MgSO₄; 2.4 CaCl₂; 26 NaHCO₃; 1.25 NaH₂PO₄; and 10 glucose), andperfused (2 ml/min) with the oxygenated ACSF in an interface chamber at30-31 ° C. Whole slices were used unless otherwise indicated. CA1pyramidal cells were recorded intracellularly (Sun, M. -K., Nelson, T.J., Xu, H., and Alkon, D. L. Calexcitin transformation of GABAergicsynapses: from excitation filter to amplifier. Proc. Natl. Acad. Sci.USA 96: 7023-7028, 1999, for cell labeling) with sharp electrodes (3MKAc; tip resistance: 60-120 M; to prevent “run-down” of GABAergicresponses in whole-cell recordings due to wash out of intracellularfactors). Stable GABAergic inhibitory postsynaptic response (IPSP) couldthus be evoked for several hours without noticeable change inamplitudes. Signals were amplified with AxoClamp-2B amplifier,digitized, stored, and analyzed using DigiData 1200 with P-Clamp6software (Axon Instruments). Frequency and amplitude values ofoscillation were taken from an average of five consecutive traces, alltriggered at the same level of the same phase. Capacitance was optimallyadjusted during discontinuous current-clamp mode before and after cellpenetration to neutralize capacitance and reduce overshoot/undershooterrors. Discontinuous single-electrode voltage-clamp mode was used forvoltage-clamping, employing a sampling rate of 3.0-5.0 kHz (30% dutycycle). Gain was usually set at 6-8 nA·mV⁻¹, slightly below the maximumvalue without causing overshoot or instability in the step response to arepetitive 10 mV step command. Bipolar stimulating electrodes(Teflon-insulated PtIr wire with 25 μm in diameter) were placed in s.pyramidale, within 200 μm, from the recording electrode, for stimulationof interneurons (50 μA, 50 μA) in the pyramidale layer. In some cases,the position of the stimulating electrodes was slightly varied withinthe CA1 cell layer to obtain monophasic postsynaptic responses. Teststimuli were applied at 1 per minute (0.017 Hz). In some experiments, anadditional stimulating electrode was placed in the stratum radiatum tostimulate the Schaffer collateral pathway (Sch). Experiments inwhich >20% variations in the evoked IPSP magnitudes occurred during the10-min control period were discarded.

Spatial maze tasks. Effects of reducing HCO₃ ⁻ formation in vivo onspatial memory were evaluated in rats with Morris watermaze task (Meiri,N., Sun, M. -K., Segal, Z., and Alkon, D. L. Memory and long-termpotentiation (LTP) dissociated: normal spatial memory despite CA1 LTPelimination with Kv1.4 antisense. Proc. Natl. Acad. Sci. USA 95:15037-15042, 1998). Male adult Wistar rats were housed in atemperature-controlled (20-24° C.) room for one week, allowed freeaccess to food and water, and kept on a 12 h light/dark cycle. On thefirst day of experiments, all rats were randomly assigned to differentgroups (10 each) and swam for 2 min in a 1.5 m (diameter)×0.6 m (depth)pool (22±1° C.). On the following day, rats were trained in a 4trial/day task for 4 consecutive days. Each training trial lasted for upto 2 min, during which rats learned to escape from water by finding ahidden platform placed in a fixed location and submerged about 1 cmbelow the water surface. A quadrant test was performed after removingthe platform, 24 h after the last training trial. The route of rats'swimming across the pool was recorded. The number of grid-crossings onrecord paper in each quadrant was counted and used as arbitrary swimmingdistance units. A single dose of ACET (5 mg/0.5 ml saline/day, freshlyprepared) was injected (intraperitoneal), about 65-70 min prior to thefirst trial or quadrant test. The control rats received the same volume(intraperitoneal) of saline.

Statistical analysis was performed using the Student's t-test for pairedor unpaired data or ANOVA whenever appropriate. The values are expressedas means±SE of the mean, with n indicating number of the cells or rats.All animals used in these experiments were treated under NationalInstitutes of Health guidelines for the welfare of laboratory animals.

Results

As seen in FIGS. 1A, B and C, carbachol (CCH)-induced θ oscillations ofhippocampal CA1 field potential and of membrane potential of CA1pyramidal cells are shown to be depend on activation of GABAergicinputs. FIGS. 1A-1, -2 and -3 contain examples of recorded fieldpotentials in hippocampal CA1: pre-CCH control (FIG. 1A-1), during CCH(50 μM, 30 min; FIG. 1A-2) and bicuculline application (BIC, 1 μM, 30min; FIG. 1A-3). FIGS. 1B-1, -2 and -3 show membrane potential traces ofrecorded CA1 pyramidal cells: pre-CCH (control; FIG. 1B1), during CCHapplication (50 μM, 30 min; the membrane was slightly depolarized andaction potential truncated; FIG. 1B2) and with membrane potentialmaintained at pre-CCH level by passing negative current; FIG. 1B3), andduring application of BIC (1 μM, 30 min; FIG. 1B4). FIG. 1C-1, -2, -3and -4 show examples of CCH-induced intracellular θ activity in 4different cells, shown at low amplification and without actionpotentials truncated.

CCH-induced θ Field Oscillation and Intracellular Theta θ RhythmActivity

To simulate cholinergic septal activation and diffuse acetylcholinetransmission (Descarries, L., Gisiger, V., and Steriade, M., Diffusetransmission by acetylcholine in the CNS, Prog. Neurobiol. 53: 603-625,1997), CCH (50 μM, 20 min), a cholinergic receptor agonist, was bathapplied to hippocampal slices from adult rats. CCH triggered a local θfield potential (FIG. 1A2; peak amplitude: 0.75±0.03 mV, mean±SE, n=12,P<0.05 from background noise; at 7.8±0.8 Hz; n=12), lasting for thepost-CCH recording period of ˜3 h (Pitler, T. A. and Alger, B. E.,Cholinergic excitation of GABAergic interneurons in the rat hippocampalslice, J. Physiol. Lond. 450: 127-142, 1992; Huerta, P. T., and Lisman,J. E., Bidirectional synaptic plasticity induced by a single burstduring cholinergic theta oscillation in CA1 in vitro, Neuron 15:1053-1063, 1995). The θ activity varied in magnitude, indicatingsummation of different numbers of neurons discharging in each phase.

The θ activity was blocked by bath atropine sulfate (1 μM, n=6; notshown), a muscarinic antagonist, as reported by others (e.g., Huerta andLisman 1995), and was generated in the CA1. The CCH-induced activity(0.73±0.04 mV, n=7, P<0.05; at 7.7±0.9 Hz; n=7, P<0.05) in CA1minislices, after dissecting away both CA3 and dentate gyrus, did notdiffer (P>0.05; unpaired t-test) from that of the whole slices. The θoscillation frequency did not change (n=8, P>0.05), although theoscillation magnitude was slightly reduced, in the presence ofkynurenate, an N-methyl-D-aspartate (NMDA)- and non-NMDA receptorantagonist (Collingridge, G. L. and Lester, R. A, Excitatory amino acidreceptors in the vertebrate central nervous system, Pharmacol. Rev. 41:14-120, 1989). Kynurenate was applied extracellularly at 500 μM (20-30min), a concentration at which it effectively abolished excitatorypostsynaptic responses of CA1 pyramidal cells to stimulation of theSchaffer collateral pathway (Sun et al. 1999) or responses of otherbrain neurons to L-glutamate (Sun, M. -K., Pharmacology ofreticulospinal vasomotor neurons in cardiovascular regulation,Pharmacol. Rev. 48: 465-494, 1996). CCH induced a θ oscillation ofmembrane potential (7.8±1.1 mV; n=20; P<0.05) in CA1 pyramidal cells(intracellular θ; FIG. 1B2); a response blocked by bath atropine sulfate(1 μM, n=8, P<0.05; not shown). At one-third to one-half of the maximumdepolarizing phase, action potentials were triggered. (FIGS. 1A2 and 1B2to 1B3). During a 5-min observing period, CCH induced an averageddischarge rate of 2.7±0.3 spikes/s, significantly higher (n=20, P<0.05)than their pre-CCH rate (0.0±0.0 spikes/s). These variations wereconsistent with those of the field θ magnitude recorded. Theintracellular θ remained unchanged when the membrane potential of thecells was maintained at their pre-CCH levels.

As shown in FIGS. 2A, B and C, carbachol (CCH)-induced θ oscillations ofhippocampal CA1 field potential and of membrane potential of CA1pyramidal cells appear to be associated with GABAergic postsynapticdepolarization. FIGS. 2A1 and 2A2 show that a single pulse stimulation(50 μA, 50 μs) of the GABAergic inputs from interneurons evokedinhibitory postsynaptic potentials (IPSPs), which were gradually reducedand reversed to depolarizing responses during CCH application (50 μM;CCH), associated with increased amplitude values of θ activity. Theaveraged maximum IPSP values of each cell during 10-min stable recordingperiod were defined as 100% baseline PSP. A minus sign was added toindicate its inhibitory nature. For clarity, only every other data pointis shown, FIGS. 2A3, 2A4 and 2A5 (calibration bars: 50 ms and 5 mV;dashed horizontal lines indicate potential level of −70 mV) showingrepresenting traces at approximate time pointed by broken arrows. FIG.2B places three of the traces together for comparison. The depolarizingresponse was blocked by BIC (1 μM, 30 min; BIC+CCH). Membrane potentialwas maintained at the pre-CCH level by passing current. FIG. 2C showsthat under voltage clamp at −74 mV, the evoked GABAergic response was anoutward current (Control), which was reversed to inward during CCHapplication (CCH, 50 μM, 20 min). Arrowheads indicate the time of thestimulation.

As seen in FIGS. 3A, B and C, carbachol shifts reversal potentials ofGABAergic postsynaptic responses in hippocampal CA1 pyramidal cells.Responses of CA1 pyramidal cells to activation of GABAergic inputs atdifferent membrane potentials before (FIG. 3A1) and in the presence ofbath 1 μM BIC, FIG. 3A2). The relationship between the maximumpostsynaptic responses and membrane potential can be described with astraight line, determined with the least sum squares criterion and wasflattened by BIC without changing the reversal potential. Responses ofCA1 pyramidal cells to activation of GABAergic inputs at differentmembrane potentials before (FIG. 3B1) and during CCH application (50 μM;FIG. 3B2). Membrane potential was maintained at the pre-CCH level bypassing current (FIG. 3B3). Arrowheads indicate the stimulation. FIG.3C1 shows an example of CCH-induced reversal of GABAergic response thatwas above threshold for generation of action potentials. Thepostsynaptic response exhibits a similar relationship between themaximum responses and membrane potential. For clarity, only 2 traces areshown. The same intensity of stimulation of the GABAergic inputstriggered an action potential in the cells post-CCH (CCH) as comparedwith IPSP pre-CCH (Control; FIG. 3C2). FIG. 3C3 shows the initialsegment at ×3 magnification with action potential truncated. Arrowheadsindicate the time when brief pulse of stimulation was delivered.

Involvement of GABAergic Postsynaptic Depolarization in the CA1 θActivities

Bath applied BIC (1 μM) eliminated the θ field oscillation (by97.5±4.2%, n=8, P<0.05; FIG. 1A2) and CA1 intracellular θ activity (by98.9±3.4%, n=10, P<0.05; FIG. 1B4). When applied before the CCHapplication, BIC did not produce obvious changes in the field potential(n=6) or membrane potentials of CA1 pyramidal cells (n=8), but preventedCCH effects on the θ activity induction. At 1 μM, BIC did not produceany obvious excitation of the CA1 cells. Activation of the GABA_(A)receptors is thus necessary for CCH to elicit synchronous CA1 fieldevents. Suppressing GABA_(A) receptor channels alone is insufficient toinduce θ.

The GABAergic inputs were activated by microstimulation of s.pyramidale. The evoked inhibitory postsynaptic potentials (IPSPs) in CA1pyramidal cells depended on the membrane potentials (e.g., FIGS. 3A1 to3A3, 3B1 to 3B3, and 3C1 to 3C3). Thus, the were always monitored withvalues compared at their pretest control membrane potentials. The evokedIPSPs (FIGS. 2A1 to 2A5; peak response: −8.89±0.29 mV, n=89) were notaltered by kynurenate (500 μM, n=6), but abolished by BIC (1 μM, by96.8±3.7%; n=8, P<0.05), indicating GABA_(A) receptor mediation and anabsence of contamination of any obvious excitatory component in theevoked IPSPs. Associated with the θ activity was a gradual reduction inthe IPSPs (n=25) and the ultimate production of an ‘excitatory’ response(FIGS. 2A1 to 2A5 and 2B; from pre-CCH −9.0±1.2 mV as compared with+5.1±0.4 mV 30 min after the CCH application; n=10, P<0.05). Thisexcitatory response was observed at the pre-CCH membrane potentialmaintained by intracellular injection of hyperpolarizing current. Thesevoltage changes in the GABAergic responses corresponded to a gradualchange of an outward current (0.18±0.03 nA) toward an inward current(0.19±0.05 nA; n=5, P<0.05) under voltage clamp (FIG. 2C). Theintracellular θ activity became evident when the GABAergic responsesbecame depolarizing (FIGS. 2A1 to 2A5). Measured when the θ activitybecame evident, the input resistance (79.2±1.6 MΩ) of the cells did notsignificantly differ (n=10; P>0.05) from their pre-CCH value (80.5±1.4MΩ). Depressing GABA_(A) responses alone was insufficient to induce theθ activity since BIC did not induce the rhythmic activity (see lastparagraph). The reversed excitatory response was also sensitive to BIC(FIG. 2B), indicating the involvement of the same type ofreceptor-channel before and after the CCH administration.

The relationship between the maximum responses of hippocampal CA1pyramidal cells to stimulation of the GABAergic inputs and membranepotential at which the inputs were activated can be described with astraight line. BIC virtually abolished the GABAergic postsynapticresponses no matter whether the postsynaptic responses were evoked atmembrane potentials positive or negative to the reversal potential(FIGS. 3A1 to 3A3). The reversal potential, however, was not changed byBIC (FIG. 3A2; −81.3±2.6 mV; n=6). This BIC effect contrasts withCCH-induced changes that were associated with a positive-shift of thereversal potential (FIGS. 3B1 to 3B3; from −79.8±3.2 to −68.4±2.8 mV;n=10, P<0.05). Thus, the CCH-induced changes in GABAergic responses arefundamentally distinct from a reduced response and could not result froma diminished GABAergic synaptic transmission (suppressed presynapticrelease or postsynaptic response). FIGS. 3C1 to 3C3 illustrate anexample in which the CCH-induced reversal potential appears to be abovethe threshold (approximately −57 mV) for generation of action potential.Thus single brief pulse of stimulation of the GABAergic inputs elicitedaction potential during post-CCH period in the cell, in contrast toinhibitory postsynaptic response before the CCH application (FIGS. 3C1to 3C3).

Elimination of CA1 θ Activities and GABA Depolarization by CarbonicAnhydrase Inhibitors

As shown in FIGS. 4A1 to 4A3, and 5B1 to 5B3, carbachol (CCH)-induced θoscillations of hippocampal CA1 field potential and of membranepotential of CA1 pyramidal cells depend on HCO₃ ⁻ formation. An exampleof recorded field potentials in hippocampal CA1: pre-CCH control (FIG.4A1), during CCH (50 μM, 30 min; FIG. 4A2) and acetazolamide (ACET)application (1 μM, 30 min; FIG. 4A3). Membrane potential traces ofrecorded CA1 pyramidal cells: pre-CCH control (4B1), during CCHapplication (50 μM, 30 min) in the presence of ACET (1 μM, 30 min; FIG.4B2). In the presence of 1 μM ACET, single pulse stimulation (50 μA, 50μs) of the GABAergic inputs evoked an IPSP (Control), which was notaltered by CCH application (50 μM, 30 min) (FIG. 4B3).

As seen in FIGS. 5A, 5B and 5C1 to 5C2, intracellular administration ofcalexcitin associated with postsynaptic depolarization inducedacetazolamide-sensitive intracellular θ in hippocampal pyramidal cells.Before the application, the membrane potential of the CA1 pyramidal celldid not show θ activity (FIG. 5A). Calexcitin application (associatedwith a depolarizing current of 0.4-0.6 nA during the off-period to evoke4-8 spikes/s to load Ca²⁺) into the recorded neuron induced theintracellular (FIG. 5B) θ. In the presence of 1 μM ACET, calexcitinapplication (associated with a depolarizing current of 0.4-0.6 nA duringthe OFF-period to evoke 4-8 spikes/s to load Ca²⁺) into the recordedneuron did not induce the intracellular theta rhythm activity θ (FIG.5C1 and 5C2).

Bath ACET (1 μM, a carbonic anhydrase inhibitor, eliminated theCCH-induced changes in GABAergic postsynaptic responses (FIG. 4B2). Theevoked IPSP (−7.7±1.0 mV, n=12, P<0.05) in the presence of ACET and CCHdid not differ (n=12, P>0.05) from their control values (−7.8±1.1 mV).Under such conditions, neither θ field oscillation (n=8; FIG. 4A) norintracellular θ activity (n=10; FIG. 4B3) was induced by CCH. Similarly,intracellular application of benzolamide, a membrane-impermeablecarbonic anhydrase inhibitor, prevented the occurrence of CCH-inducedreversed GABAergic responses and intracellular θ activity (n=6),indicating an involvement of intracellular carbonic anhydrase.Interestingly, application of calexcitin, a memory-related signalprotein (Alkon, D. L., Nelson, T. J., Zhao, W. Q., and Cavallaro, S.,Time domains of neuronal Ca²⁺ signaling and associative memory: stepsthrough a calexcitin, ryanodine receptor, K⁺ channel cascade, TrendsNeurosci. 21: 529-537, 1998; Sun et al. 1999), into CA1 pyramidal cellsmimicked CCH in inducing the intracellular θ activity (FIG. 5B; n=10),when associated with a depolarizing current to load Ca²⁺. Thecalexcitin-induced intracellular θ activity was also prevented by bathACET (1 μM) in six cells tested (FIG. 5C2). These results indicate acritical role of HCO₃ ⁻ conductance in an intracellular signalingcascade responsible for the θ rhythm.

Entraining CA1 Pyramidal Cells by GABAergic Inputs

As shown in FIGS. 6A, B, C and D, rebound action potentials ofhippocampal CA1 pyramidal cells evoked by GABAergic inhibition vary inoccurrence and timing. In slowly adapting cells, an evoked IPSP candelay or reset the subsequent occurrence of spikes when the cells weredepolarized (FIG. 6A). The vertical lines above the trace indicate theexpected time for an action potential to occur if the cell continued todischarge at the same regular intervals observed immediately before thestimulus was delivered. Arrowhead indicates the stimulation. Rebounddepolarization was not evoked at resting membrane potential with singlepulse (trace 1) or a train of 4 pulses at 100 Hz (trace 2) stimulationof the GABAergic inputs (FIG. 6B). Rebound action potential at restingmembrane potential requires too strong hyperpolarization (FIG. 6C) (≧30mV; trace 3 with action potential truncated), otherwise no rebounddepolarization was evoked (trace 1). When depolarized, rebound actionpotential can be induced but with low safety and varied timing (withaction potential truncated) (FIG. 6D).

As seen in FIGS. 7A1 to 7A3, 7B1 to 7B2 and 7C1 to 7C5, carbachol(CCH)-induced θ GABAergic depolarization of hippocampal CA1 pyramidalcells enables GABAergic inputs to entrain CA1 pyramidal cells. θ rhythmstimulation evoked IPSPs before CCH application (Control; FIG. 7A1).During CCH application (50 μM, 30 min; FIG. 7A2), the same pattern ofstimulation entrained activity of the pyramidal cell. Action potentialswere evoked at the 2nd, 3rd, and 5th pulses, with action potentialstruncated. The evoked postsynaptic responses were abolished by BIC (1μM, 30 min; FIG. 7A3). A schematic diagram of the supposed network anddischarge relationship between CA1 pyramidal cells and GABAergicinterneurons is shown in FIG. 7B1. Cholinergic inputs (synaptic ordiffuse transmission) act on pyramidal cells, inducing HCO₃ ⁻accumulation and enhance HCO₃ ⁻ conductance through the GABA_(A)receptor channels. The θ rhythmic activity of GABAergic interneurons canthen directly be transmitted to the pyramidal cells, entraining theiractivity and altering signal processing. FIG. 7B2 models a peakdischarge relationship of pyramidal cells (black rectangle) andinterneurons (shadow oval) in θ rhythm in behaving and rapid eyemovement (REM) sleep (based on O'Keefe and Recce 1993; Shen et al. 1997;Csicsvari et al. 1999). The arrow indicates the discharge shift of aplace cell, starting from shadow rectangle, in relation to the θactivity as the animal travels into the place field of the place cell.GABA, GABAergic interneurons; Pyr, CA1 pyramidal cells; SCH, Schaffercollateral pathway. FIGS. 7C1 to 7C3 show examples of traces withouttruncation, showing that the brief pulse of stimulation at 5.5 Hzelicited action potentials even though the 1st brief pulse ofstimulation was insufficient to evoke action potential. FIG. 7C4illustrates responses to co-stimulation of SCH at below-thresholdintensity and GABAergic inputs at pre-CCH (Control) and post-CCH (CCH)periods. FIG. 7C5 shows the initial segment at ×3 amplification withaction potential truncated. Arrowheads indicate the time when the briefpulse of stimulation was delivered.

Entraining hippocampal pyramidal cells at θ frequency has been proposedto be a fundamental role of the interneurons (Cobb, S. R., Buhl, E. H.,Halasy, K., Paulsen, O., and Somogyi, P., Synchronization of neuronalactivity in hippocampus by individual GABAergic interneurons, Nature378: 75-78, 1995; Paulsen, O. and Moser, E. I., A model of hippocampalmemory encoding and retrieval: GABAergic control of synaptic plasticity,Trends Neurosci. 21: 273-278, 1998). The only previously proposedmechanism for how GABAergic interneurons entrain the pyramidal cells isrebound action potential. However, rebound ‘depolarization’ usuallyrequires resting activity that was provided by constant currentinjection (Cobb et al. 1995) and hippocampal pyramidal cells normally donot show much spontaneous activity. In some cells (26 out of 149 neuronsin which effects of membrane potential changes on the GABAergicpostsynaptic responses were examined), discharges lasted for a period ofelicited depolarization and an evoked IPSP appeared to be able to delaysubsequent spikes (FIG. 6A). The majority of cells (123 out of 149),however, showed a rapid adaptation to depolarization (FIG. 3A1 to 3A3and 3B1 to 3B3), resulting in a silent but depolarized state. At restingmembrane potential, rebound depolarization requires very stronghyperpolarization, which naturally occurring IPSPs are unlikely toprovide. No rebound action potential was observed with IPSPs of −8.9±0.3mV evoked at resting membrane potentials (−73.8±0.9 mV, n=89; FIG. 6B,trace 1). A train of pulses at 100 Hz was also ineffective (FIG. 6B,trace 2), suggesting that temporal summation of the unitary IPSPs isinsufficient to evoke rebound depolarization. Furthermore, nosignificant rebound depolarization (0.19±0.12 mV, n=75, P>0.05) wasevoked with intracellular pulses (up to 700 ms) sufficient to evoke−10.8±1.4 mV potential changes (FIG. 6C, trace 1) from their restingmembrane potential (−74.8±0.4 mV). In addition, when evoked atdepolarized membrane potentials, the occurrence and timing of individual‘rebound’ action potentials varied (FIG. 6D). Thus, rebound actionpotentials, even when they occur, do not represent a precise controlmechanism. On the other hand, in the presence of CCH, stimulation ofGABAergic inputs elicited instantly phase-locked firing of pyramidalcells (FIGS. 7A2 and 7C1 to 7C3; n=14). The postsynaptic GABAergicresponse to the first stimulation pulse usually did not reach actionpotential threshold (FIGS. 7A2 and 7C1 to 7C3). The postsynapticGABAergic responses were sensitive to BIC, indicating the involvement ofthe same receptor-channels (FIGS. 7A3). In eight cells, single pulsestimulation of SCH (10-30 μA, 50 μs) evoked an excitatory postsynapticpotential of 7.5±1.2 mV, which was about 50% below the threshold. Beforethe CCH administration, co-stimulation of SCH at the set intensity (50%below the threshold) and GABAergic inputs (50 μA, 50 μs) largelyabolished the SCH stimulation-induced excitatory potential (by89.5±4.3%, n=8, P<0.05; FIG. 7C4). The single-pulse SCHstimulation-evoked excitatory postsynaptic potential was not altered(P>0.05) by CCH (not shown). Action potentials, however, were evoked byco-stimulation of Sch at below-threshold intensity together withreversed GABAergic inputs in all cases (n=8, P<0.05; FIG. 7C-4). Thus,reversed synaptic responses reshapes the GABAergic inhibitory functioninto amplification (Sun et al. 1999) and reconfigures the operations ofhippocampal networks into patterns of activity associated with GABAergicinputs (FIG. 7B1 and 7B2).

Spatial Memory Deficits by ACET Administration in vivo

As shown in FIGS. 8A, 8B, 8C1 to 8C2, 8D1 to 8D2 and 8E, carbonicanhydrase inhibitors impair rat spatial memory in vivo. FIG. 8A showsthe mean (±SE) escape latency across 16 trials (F_(15,270)=22.93,P<0.0001) in the watermaze by rats given a single dose (indicated witharrows) of saline (Saline, 0.5 ml) or ACET (5 mg/0.5 ml/day ip). FIG. 8Billustrates the percentage ratio in escape latency of the 1^(st) trialof the day between the two groups. FIGS. 8C-8E show the quadrantpreference of saline-(n=10; ** P<0.0001; 8C1) and ACET-injected rats(n=10; FIG. 8D1) and swimming distance (in 1 min; FIG. 8E). A platformfor escape was placed in quadrant 4 during training. FIGS. 8C2 and 8D2show paths taken by representative rats with quadrant numbers indicated.

As seen in FIGS. 9A, B and C, ACET administration does not affectretrieval of formed spatial memory. FIG. 9A shows the escape latency ofthe control rats during three more days of training trials. FIGS. 9B1and 9C1 illustrate quadrant preference of these rats after a single doseof saline (0.5 ml, n=5; FIG. 9B1) or ACET (5 mg/0.5 ml, n=5; FIG. 9C1).No significant difference in quadrant preference (P>0.05) was observedbetween the saline- and ACET-injected rats. FIGS. 9B2 and 9C2 show pathstaken by representative rats with quadrant numbers indicated.

Reducing HCO₃ ⁻ formation with a carbonic anhydrase inhibitor that canpass through the blood brain barrier can affect rat spatial memory. Inrats, an intraperitoneal (ip) dose of ACET produces a peak concentrationin the blood within 1 hr and is cleared by 2 h (Cassin, S., Beck, M. J.,Travis, P., Sanders, S., and Otis, A. B., The Effect of CarbonicAnhydrase Inhibition in Exercising Rats, Brooks Air Force Base, TX: USAir Force School of Aerospace Medicine, 1963, pp. 1-6; Sone, M., Sei,H., Morita, Y., Ogura, T., and Sone, S., The effects of acetazolamide onarterial pressure variability during REM sleep in the rat, Physiol.Behav. 63: 213-218, 1998). Effects of ACET on spatial learning (Meiri etal. 1998) were determined during this short period.

A single dose of ACET (14-18 mg/kg, sufficient to reduce the EEG θ powerby about 50% at maximum during rat REM sleep; Sone et al. 1998) wassufficient to produce memory impairment (FIG. 8A). The ACET group showeda strikingly smaller reduction (F_(1,18)=34.79, P<0.0001) in escapelatency during training trials than the saline group did. The memoryimpairment became more significant as the training days progressed andwas particularly evident in the first trial (65-70 min after theinjection) of each successive day (FIGS. 8A and B). The latter mightreflect a relatively normal short-term (vs. long-term) learning afterACET or more likely influence of a rapid clearance of the drug (Cassinet al. 1963; Sone et al. 1998). Quadrant tests 24 h after the lasttraining trial revealed that control rats spent the majority of theirtime searching in the quadrant (Quadrant 4; FIG. 8C1) where the platformwas previously placed and had been removed (F_(3,36)=183.9, P<0.0001;ANOVA and Newman-Keuls post hoc test), whereas the ACET group showed nopreference to a particular quadrant (F_(3,36)=1.59, P=0.21; FIG. 8D1).

The total swimming distances, however, did not differ between the twogroups (FIG. 8E; P>0.05), indicating that ACET did not grossly affecttheir sensory or locomotor activities. Neither was memory retrievalaffected by ACET. The control rats were trained for 3 more days (FIG.9A) and received the single injection of either ACET or saline 24 hafter the last training trial. Sixty-five to seventy min after theinjection, a quadrant test in ACET-injected rats showed no significantdifference (P>0.05) in quadrant 4 preference (F_(3,16)=132.9, P<0.0001;FIG. 9C) from that of the saline control rats (F_(3,16)=306.4, P<0.0001;FIG. 9B). These results indicate that once formed, memory and itsrecall, as well as the sensory stimuli that elicit recall, are notvulnerable to ACET. During the experimental periods, no rats showed anyapparent sign of discomfort or abnormal behaviors such as hypo- orhyperactivity.

Discussion

In vitro θ Rhythm and Cholinergic Involvement

The CCH-induced θ of this experiment is consistent with the in vitro θpreviously reported by many other groups (e.g., Golebiewski H,Eckersdorf B, and Konopacki J., Cholinergic/GABAergic interaction in theproduction of EEG theta oscillations in rat hippocampal formation invitro., Acta Neurobiol Exp 56: 147-153, 1996; Konopacki J, andGolebiewski H., Theta-like activity in hippocampal formation slices:cholinergic-GABAergic interaction., NeuroReport 4: 963-966, 1993;Huertaand Lisman 1995; Vertes and Kocsis 1997) and appears to be fundamentallyidentical to the θ rhythm in vivo for its sensitivity to muscarinicreceptor antagonists, dependence on GABAergic interneurons, andindependence of glutamatergic inputs. Acetylcholine's activation ofmuscarinic receptors on pyramidal cells is considered to be modulatoryand much too slow to generate rhythmic θ directly (Dutar et al. 1995;Vertes and Kocsis 1997). The ineffectiveness of blocking glutamatergicinputs on the θ is also consistent with the evidence that during eoscillations in vivo CA3 neurons rarely reach action potential threshold(Bland, B. H. and Wishaw, I. Q., Generators and topography ofhippocampal theta (RSA) in the anaesthetized and freely moving rat,Brain. Res. 118: 259-280, 1976; Fox, S. E. and Ranck, J. B. Jr.,Electrophysiological characteristics of hippocampal complex-spike cellsand theta cells, Exp. Brain. Res. 41: 399-410, 1981) and excitatoryinputs from CA3 are unlikely to contribute to CA1 θ (Thompson, L. T. andBest, P. J., Place cells and silent cells in the hippocampus offreely-behaving rats, J. Neurosci. 9: 2382-2390, 1989; Soltesz andDeschenes 1993; Ylinen et al. 1995). The effectiveness of the specificGABA_(A) receptor antagonist BIC in eliminating the postsynapticresponse and θ activity strongly suggests that GABA_(B) receptoractivation did not contribute significantly to the responses. The CA1θactivity does, however, appear to be distinct from the activityoscillations that were BIC-insensitive, involved epileptiform bursting,and were generated by CA3 neurons in one report (Williams, J. H. andKauer, J. A., Properties of carbachol-induced oscillatory activity inrat hippocampus, J. Neurophysiol. 78: 2631-2640, 1997). This differencemay depend on the preparations or age of the animals. In their study,slices were obtained from younger animals so that cells may have a highintracellular Cl⁻ concentration, due to the lack of a developmentallyexpressed Cl⁻-extruding K⁺/Cl⁻ co-transporter in early age (Rivera, C.,Voipio, J., Payne, J. A., Ruusuvuori, E., Lahtinen, H., Lamsa, K.,Pirvola, U., Saarma, M., and Kaila, K., The K⁺/Cl⁻ co-transporter KCC2renders GABA hyperpolarizing during neuronal maturation, Nature 397:251-255, 1999).

Involvement of muscarinic receptors in hippocampal θ induction has beenwell established. Low-frequency MS/DBv stimulation activates cholinergicinputs to the hippocampus and drives θ in vivo (Descarries et al. 1997).Microinfuisions of CCH or eserine into areas including CA1 induce anatropine-sensitive hippocampal θ activity in vivo (Rowntree, C. I. andBland, B. H., An analysis of cholinoceptive neurons in the hippocampalformation by direct microinfusion, Brain Res. 362: 98-113, 1986).Atropine administration has been found to eliminate hippocampal θ invivo (Brazhnik, E. S. and Vinogradova, O. S., Control of the neuronalrhythmic bursts in the septal pacemaker of theta-rhythm: effects ofanaesthetic and antichlinergic drugs, Brain Res. 380: 94-106, 1986;Vertes and Kocsis 1997). The effectiveness of muscarinic antagonistsdoes not mean, however, that there is only one form of θ. Inanesthetized rats, atropine eliminates θ (Stewart, M. and Fox, S. E.,Detection of an atropine-resistant component of the hippocampal thetarhythm in urethane-anesthetized rats, Brain Res. 500: 55-60, 1989).Under such conditions, an unconventional small “residual θ” wasdescribed, that, in the absence of θ, could be shown by using MS/Devneurons that discharged rhythmically to trigger hippocampal EEG inanalysis (Stewart and Fox 1989). The involvement of serotonergictransmission has been proposed (Vanderwolf, C. H., Harvey, G. C., andLeung, L. W., Transcallosal evoked potentials in relation to behavior inthe rat: effects of atropine, p-chloropheylalanine, reserpine,scopolamine and trifluoperazine, Behav. Brain Res. 25: 31-48, 1987). Theparticular role and importance of such an atropine-resistant componentin memory remains to be established. Furthermore, intracellular θactivity of pyramidal cells has been claimed to result from depolarizingor hyperpolarizing membrane potential oscillations (Vertes and Kocsis1997).

The θ activities induced in here were most likely evoked by muscarinicreceptor activation, given their sensitivity to atropine. However, it ispossible that multiple cell targets might be required for CCH to inducethe θ activity. Although, it should not be ruled out entirely, nicotinicreceptors are probably not involved because the interneurons in or nearthe stratum pyramidale and with axonal projections within and aroundthis layer exhibit no nicotinic response (McQuiston, A. R. and Madison,D. V., Nicotinic receptor activation excites distinct subtypes ofinterneurons in the rat hippocampus, J. Neurosci. 19: 2887-2896, 1999).

HCO3⁻-Mediated GABAergic Synaptic Depolarization

Encoding experiences into lasting memory may involve a qualitativediversity of synaptic plasticity (Otis, T., Zhang, S., and Trussell, L.O., Direct measurement of AMPA receptor desensitization induced byglutamatergic synaptic transmission, J. Neurosci. 16: 7496-7504, 1986;Kornhauser, J. M. and Greenberg, M. E., A kinase to remember: dual rolesfor MAP kinase in long-term memory, Neuron 18: 839-842, 1997; Brenowitz,S., David, J., and Trussell, L., Enhancement of synaptic efficacy bypresynaptic GABA(B) receptor, Neuron 20: 135-141, 1998; Paulsen andMoser 1998), including changing operations of preexisting synapses andgrowing new ones. GABAergic postsynaptic depolarizing responses havebeen observed by several groups (Michelson, H. B. and Wong, P. K. S.,Excitatory synaptic responses mediated by GABA_(A) receptors in thehippocampus, Science 253: 1420-1423, 1991; Alkon, D. L., Sanchez-Andres,J. -V., Ito, E., Oka, K., Yoshioka, T., and Collin, C., Long-termtransformation of an inhibitory into an excitatory GABAergic synapticresponse, Proc. Natl. Acad. Sci. USA 89: 11862-11866, 1992; Kaila, K.,Voipio, J., Paalasmaa, P., Paternack, M., and Deisz, R. A., The role ofbicarbonate in GABA_(A) receptor-mediated IPSPs of rat neocorticalneurones, J. Physiol. Lond. 464: 273-289, 1993; Siklós, L., Rickmann,M., Joó, F., Freeman, W. J., and Wolff, J. R., Chloride ispreferentially accumulated in a subpopulation of dendrites andperiglomerular cells of the main olfactory bulb in adult rats,Neuroscience 64: 165-172, 1995; Staley, K. J., Soldo, B. L., andProctor, W. R., Ionic mechanisms of neuronal excitation by inhibitoryGABA_(A) receptors, Science 269: 977-981, 1995; Rivera et al. 1999). Thedepolarization induced in the present study differs from that reportedby Kalia et al. (Kalia, K., Lamsa, K., Smimov, S., Taira, S., andVoipio, J. Long-lasting GABA-mediated depolarization evoked byhigh-frequency stimulation in pyramidal neurons of rat hippocampal sliceis attributable to a network-driven, bicarbonate-dependent K⁺ transient.J. Neurosci. 17: 7662-7672, 1997), who applied a high-frequency train ofpulses to the stratum radiatum to induce depolarizing responses thatshowed a slow time course but lasted for several seconds. Nevertheless,these experimental results are consistent with the evidence thatGABAergic depolarization can be induced by enhancing HCO₃ ⁻ conductancethrough GABA_(A) receptor-channels in adult hippocampal cells, aresponse sensitive to carbonic anhydrase inhibitors (Kaila et al. 1993;Staley et al. 1995). Carbonic anhydrase exists in pyramidal cells(Pasternack, M., Voipio, J., and Kaila, K. Intracellular carbonicanhydrase activity and its role in GABA-induced acidosis in isolated rathippocampal pyramidal neurones. Acta Physiol. Scand. 148: 229-231,1993). Indeed, the θ activity and the reversed GABAergic postsynapticresponses were largely abolished by carbonic anhydrase inhibitors. Theeffectiveness of intracellular benzolamide, a membrane-impermeablecarbonic anhydrase inhibitor, indicates that the response depends onactivity of an intracellular enzyme. Supporting the functionalimportance of carbonic anhydrase activity in synaptic plasticity is alsothe result that a partial blockade of the enzyme activity in vivomarkedly impaired retention of rat watermaze learning. HCO₃ ⁻ has areversal potential about −12 mV (Staley et al. 1995). With an increasedHCO₃ ⁻/Cl⁻ permeability ratio, outward HCO₃ ⁻ flux would depolarize themembrane at the resting membrane potential. Alteration in HCO₃ ⁻conductance and/or transmembrane concentrations (thus the driving force)would be expected to dramatically alter the synaptic response.

The existence of physiological regulators of carbonic anhydrase has beenproposed in other contexts, including those that activate the anhydraseby facilitating its membrane association (Parkes, J. L. and Coleman, P.S., Enhancement of carbonic anhydrase activity by erythrocytemembranes., Arch. Biochem. Biophys., 275: 459-468, 1989). In molluscanneurons, the carbonic anhydrase-HCO₃ ⁻ system has been found to be themost potent regulatory factor in intracellular pH regulation.Depolarized snail neurons, for example, were associated with increasedproton conductance (e.g., Thomas R C, and Meech R W., Hydrogen ioncurrents and intracellular pH in depolarized voltage-clamped snailneurones., Nature 299: 826-828, 1982). Changes in intracellular pH couldalso alter ion channel function as well as metabolic activity. Itremains to be determined whether intracellular pH is significantlyaltered or plays a role in the CCH-induced θ and/or regulation of memorybehavior.

Phase Relationship of θ Activities of CA1 Pyramidal Cells andInterneurons

Two major classes of hippocampal CA1 neurons are the θ cells and the“place cells” (Paulsen and Moser 1998). The GABAergic interneurons,including basket cells and axoaxonic cells, have been called θ cells(Paulsen and Moser 1998). The basket interneurons are particularlyactive and express strongest rhythmic discharges (Cscsvari et al. 1999)when hippocampal EEG is dominated by θ rhythm. One basket interneuronselectively and perisomatically innervates approximately 1,000 pyramidalcells (Cobb et al. 1995) and thus can entrain a large population. Thepyramidal neurons, on the other hand, are largely quiescent during θrhythm associated with exploration, but a subpopulation shows strongfiring that is highly correlated with specific locations in space (Dutaret al. 1995; Vertes and Kocsis 1997; Paulsen and Moser 1998). These“place” cells fire at all phases of the θ rhythm (O'Keefe and Recce1993). Here, the results show that during θ oscillation, the GABAergicpostsynaptic responses are altered. Gating through a postsynapticmechanism, as described in the present study, could explain why somepyramidal cells become active while the vast majority of others remainsilent during θ EEG, even if they are innervated by the sameinterneuron.

Every pyramidal cell is innervated by 10-12 GABAergic interneurons,preferentially making synapses on cell bodies, proximal dendrites, andaxon initial segments of CA1 pyramidal cells (Buhl E H, Halasy K, andSomogyi P., Diverse sources of hippocampal unitary inhibitorypostsynaptic potentials and the number of synaptic release sites.,Nature 368: 823-828, 1994; Paulsen and Moser 1998 for review). Ifpyramidal cells were activated by rebound excitation from GABAergicinhibition, one would expect that pyramidal cells should discharge wheninterneurons become silent. This may be the case in anesthetized states.The intracellular θ activity of CA1 pyramidal cells when recorded underanesthesia have often been reported to fire out-of-phase, delayed abouta half cycle (Soltesz and Deschenes 1993; Ylinen et al. 1995). Inbehaving animals or during REM sleep, however, the earlier dischargepeaks of these interneurons precede peaks of population activity ofpyramidal cells during θ activity and both pyramidal cell firing andinterneuronal discharge occur within the same θ phase period (Cscsvariet al. 1999). Anesthesia is known to attenuate a large peak of θ,revealing rhythmic hyperpolarization of pyramidal cells from basketinterneurons (Ylinen et al. 1995). Thus, the θ activity duringexploration or induced by cholinergic agonists in vitro seemsincomparable to the θ under anesthesia (Ylinen et al. 1995; Muir, G. M.and Bilkey, D. K. Synchronous modulation of perirhinal cortex neuronalactivity during cholinergically mediated (type II) hippocampal theta.Hippocampus 8: 526-532, 1998).

Not only does the discharge phase relationship between pyramidal cellsand interneurons differ between the anesthetized and behaving animals,but the phase relationship is also dynamic. In behaving animals, thephase forward shift of the discharges of place cells on each θ cycleoccurs during traversal of the place field of the cell (O'Keefe andRecce 1993; Shen et al. 1997). Place cells thus fire in phase withprogressively stronger GABAergic inputs from interneurons and at earlierphases of the θ cycles as the rat moves toward the center of their placefield (FIG. 7B; O'Keefe and Recce 1993; Shen et al. 1997; Csicsvari etal. 1999). Mechanism(s) responsible for the θ initiation or entrainingof the pyramidal cell activity should be able to code for timing(Trussell, L. O. Synaptic mechanisms for coding timing in auditoryneurons. Annu. Rev. Physiol. 61: 477-496, 1999) or entrain pyramidalcells at different θ phases, including those with minimal delay(Csicsvari et al. 1999). A rebound excitation followinghyperpolarization is unlikely to have such multi-phase capability as theinterneurons become more active during θ-related activity. A briefswitch toward or to GABAergic depolarization would be more effective andreliable in processing dynamic information. Strong GABAergic inputsafter the synaptic switch can entrain the activity of pyramidal cells sothat the delay would be relatively short and evoke an “in phase”activity.

The present results suggest that the GABAergic entraining could resultin three ways. In a small percentage of cells, CCH was able to elevatethe reversal potential to levels that were above the threshold for spikeactivity. GABAergic inputs thus could directly drive, even if briefly,activity of the pyramidal cells with sufficient transformation ofhyperpolarizing to depolarizing responses. The reversed response,although often not strong enough to reach threshold by itself, canentrain the pyramidal cells when stimulated at a θ frequency (FIGS. 7A1to 7A3, 7B1 to 7B2, and 7C1 to 7C5). Furthermore, the reversed responsecan effectively enhance weak excitatory inputs to reach threshold (FIGS.7C1 to 7C5) (Sun et al. 1999). When the inputs are not very strong andrequire summation of multiple synaptic activation or other associativeinputs, such as glutamatergic inputs (Sun et al. 1999) to reach thethreshold, the entrained action potentials are likely to be delayed. Itshould also be noted that for this summation effect to occur, there mustbe sufficient spatial proximity on the pyramidal cells for theglutamatergic excitatory postsynaptic potentials to spread from thedendrites to the somata where they would interact with the transformedGABAergic IPSPs. Thus, “place cells” are capable of firing at all phasesof the θ rhythm in relation to the activity of GABAergic interneurons.

This Example shows reversed, HCO₃ ⁻-dependent GABAergic postsynapticresponses and their effectiveness in entraining activity of pyramidalcells. The most reasonable explanation for our results is an essentialrequirement for carbonic anhydrase activity in the molecular signalingpathways, for learning and memory. This result surprisingly offers anexplanation for the occurrence of mental retardation in carbonicanhydrase II-deficient patients (Sly, W. S. and Hu, P. Y. Human carbonicanhydrases and carbonic anhydrase deficiencies. Annu. Rev. Biochem. 64:375-401, 1995). Carbonic anhydrase is very efficient and may act as afunctional switch. The effectiveness of inhibiting carbonic anhydrase toimpair rat spatial memory was not predicted by evidence ofbicarbonate-dependent GABAergic depolarization, as defined in vitro.That is spatial memory is a much more complex phenomenon thandepolarization. The critical role of HCO₃ ⁻ in θ activity alsosurprisingly explains the fact that ACET is effective in the treatmentof central sleep apnea or epilepsy, causing somnolence together withsignificant decreases in centrally originated θ rhythm-relatedfluctuations in cardiorespiratory functions (Sone et al. 1998).ACET-regulated HCO₃ ⁻ gradients appear important for acquisition ofmemory rather than retrieval from formed memory. Such compounds may haveclinical value when temporarily suppressed memory is beneficial (e.g.,in surgery or post-traumatic-stress-disorder).

Example 2

Encoding an experience into a lasting memory is thought to involve analtered operation of relevant synapses and a variety of othersubcellular processes, including changed activity of specific proteins.The following example demonstrates that co-applying (associating)membrane depolarization of rat hippocampal CA1 pyramidal cells withintracellular microinjections of calexcitin (CE), a memory-relatedsignaling protein, induces a long-term transformation of inhibitorypostsynaptic potentials from basket interneurons (BAS) into excitatorypostsynaptic potentials. This synaptic transformation changes thefunction of the synaptic inputs from excitation filter to amplifier, isaccompanied by a shift of the reversal potential of BAS-CA1 postsynapticpotentials, and is blocked by inhibiting carbonic anhydrase orantagonizing ryanodine receptors. Effects in the opposite direction areproduced when anti-CE antibody is introduced into the cells, whereasheat-inactivated CE and antibodies are ineffective. These data suggestthat CE is actively involved in shaping BAS-CA1 synaptic plasticity andcontrolling information processing through the hippocampal networks.

Introduction

Synapses are considered a critical site at final targets through whichmemory-related events realize their functional expression (Kornhauser,J. M. & Greenberg, M. E. (1997) Neuron 18, 839-842), whether the eventsinvolve changed gene expression and protein translation, altered kinaseactivities, or modified signaling cascades. A few proteins have beenimplicated in associative memory. These include Ca²⁺/calmodulin IIkinases, protein kinase C (PKC), and calexcitin (CE), a recently clonedand sequenced 22-kDa learning-associated Ca²⁺-binding protein (Alkon, D.L., Nelson, T. J., Zhao, W. Q. & Cavallaro, S. (1998) Trends Neurosci.21, 529-537; Nelson, T. J., Collin, C. & Alkon, D. L. (1990) Science247, 1479-1483), and the type II ryanodine receptors (RyR). Levels of CEin identified mollusk neurons change with Pavlovian conditioning (Nelsonet al. 1990). CE is also a substrate of PKC and may play a role inpathophysiology of Alzheimer disease (Alkon et al. 1998). It increasesneuronal excitability in Hermissenda and mammalian hippocampus andcerebellum, whereas anti-CE antibodies react with 22-kDa proteinfractions from mammalian brain extracts (Alkon et al. 1998).Furthermore, biochemical and patch-clamp studies indicate that CEactivates the RyR to release intracellular Ca²⁺ from the endoplasmicreticulum (Alkon et al. 1998). These functional similarities in diversespecies suggest homologous protein targets and mechanistic conservationacross evolution.

Abbreviations

ACET, acetazolamide; BAPTA,1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; BAS, basketneurons; CA, carbonic anhydrase; CE, calexcitin; EPSPs, excitatorypostsynaptic potentials; GABA, γ-aminobutyrate; IPSPs, inhibitorypostsynaptic potentials; KYN, kynurenate; PSP, postsynaptic potential;RR, ruthenium red; RyR, ryanodine receptors; SCH, Schaffer collateralpathway.

Methods

Chemicals. Cloned CE (without the C-terminal P-loop) containing a His₉leader sequence was expressed in BL21 (DE3) Escherichia coli cells andpurified by repeated affinity chromatography on Ni²⁺-charged His-Bindcolumns (Novagen). Anti-CE antibody was raised in rabbits, by usingpeptide Ac-DVNDTSGDNIIDKHEYSTC-NH, corresponding to positions 115-133 ofCE, conjugated to keyhole limpet hemocyanin(KLH; linked via C-terminalcysteine). The antibody was effective against nondenatured CE only.Agents were either injected into the recorded cells through therecording electrodes: CE, anti-CE antibody, ruthenium red (RR), or1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA); orinto the perfusion medium: kynurenic acid (KYN; Sigma; 500 μM; adjustedto pH 7.4 with 1 M NaOH; Sun, M. -K. (1996) Pharmacol. Rev. 48,465-494), bicuculline methiodide (Sigma; 10 μM), acetazolamide (ACET;Sigma; 1 or 10 μM), or benzolamide (gift from T. H. Maren, University ofFlorida, Gainesville). For injections of the proteins, electrode tipswere filled with 1 μl (260 ng/μl) of cloned CE, heat-inactivated CE, oranti-CE antibody or heat-inactivated anti-CE antibody, respectively, in1 M potassium acetate (KOAc) and backfilled with 3 M KOAc (pH adjustedto 7.25). The proteins, BAPTA (˜10 mM), and RR were injected duringpulse cycles controlled with PCLAMP program (the proteins and BAPTA:−2.0 nA, 700 ms on 33% duty cycles for 15 min; RR: +0.5 nA, 500 ms 50%on duty cycles through 2 mM solution for 10 min). Heat-inactivatedproteins (100° C. for 5 min) were used as control. For carbonicanhydrase (CA) activity measurement, a 1-μl sample of purified CA(Sigma) and rat brain homogenate in 1 ml of 0.1 M Tris-HCl (pH 7.4) wasbubbled at 4° C. with CO₂ from a gas cylinder. pH changes were monitoredwith an Orion 9802 BH pH electrode connected to a data acquisitionsystem via a VWR 8010 pH meter.

Electrophysiology. Male Sprague-Dawley rats (130-180 g) were decapitatedand the brains were removed and cooled rapidly in a modified artificialcerebrospinal fluid (aCSF) (about 4° C.), bubbled continuously with 95%O₂/5% CO₂. Hippocampi were sliced (400 μm), placed in oxygenated aCSF(124 mM NaCl/3 mM KCl/1.3 mM MgSO₄/2.4 mM CaCl₂/26 mM NaHCO₃/1.25 mMNaH₂PO₄/10 mM glucose), and subfused (2 ml/min) with the oxygenated aCSFin an interface chamber and allowed to equilibrate for a minimum of 1 hrat 30-31° C. Hepes was used to replace NaHCO₃ in non-bicarbonate buffersolution, which was bubbled with 100% O₂ (pH adjusted to 7.38-7.40). CA1pyramidal cells were recorded intracellularly with sharp electrodes.Intracellular microelectrode recording rather than whole-cell clampavoids immediate internal perfusion of the test proteins and agents intothe cells and marked run-down of γ-aminobutyrate (GABA)-evoked currents.A control period without immediate influence of test proteins is crucialfor evaluating test results. KOAc (3 M, pH 7.25)-filled electrodes (tipresistance 60-120 MΩ) were positioned in the area of CA1. A bipolarstimulating electrode (Teflon-insulated PtIr wire 25 μm in diameter) wasalso placed in the stratum pyramidale, within 200 μm from the recordingelectrode, for stimulation of basket interneurons (BAS) (50 μA). In someexperiments, an additional bipolar electrode was placed in the stratumradiatum to stimulate the Schaffer collateral pathway (SCH). CA1 neuronswith stable resting membrane potential more negative than −70 mV werestudied. Unless otherwise mentioned, test stimuli were applied atfrequency of 1 per minute (0.017 Hz). Signals were amplified withAxoClamp-2B amplifier and digitized and stored by using a DigiData 1200with the PCLAMP6 data collection and analysis software (AxonInstruments) and a Pentium PC computer. Experiments in which >20%variations in the evoked inhibitory postsynaptic potentials (IPSPs)during a 10 min control period occurred were discarded. Percent baselinePSP at each minute was calculated by dividing its value by baseline PSPthen multiplying the result by 100. Baseline PSP was the mean of 10 minbefore treatments in each cell. A negative sign was added to indicateits inhibitory nature so that −100% is baseline IPSP and a positivevalue indicates an excitatory response. Differences were consideredsignificant at P<0.05.

Results

As seen in FIGS. 10A-O, CE transforms BAS-CA1 synapses. Bicuculline(BIC, 1 μM, 30 min) eliminates (FIG. 10A), whereas KYN (500 μM, 20 min)does not alter (FIG. 10B), the evoked IPSPs. The relationship betweenthe evoked BAS-CA1 PSP at different membrane potentials (MPs) (FIGS. 10Cand 10D) in a CA1 pyramidal cell can be described with a straight line(FIG. 10E), determined by the least sum squares criterion, and is notaltered by KYN (FIGS. 10C and D). CE reduces BAS-CA1 IPSP (FIG. 10F; twooverlapping traces) and shifts the PSP-MP curve to the right (FIG. 10H).Heat-inactivated CE (dN-Calexcitin) is ineffective (FIG. 10G).Microinjections of CE conjugated with the green fluorescent Alexa488(Molecular Probes) results in strong labeling of the cell body andportion of the dendrites in focus (FIGS. 10I-1, active form, and 10I-2,heat-inactivated; after fixation with 10% paraformaldehyde/salineovernight and cutting to 40 μm thick, shown ×400), indicating theefficacy of the CE microinjection. In FIG. 10J, time courses of theresponse to CE or heat-inactivated CE injection (Control), each pointrepresents the mean IPSP magnitudes+SEM normalized to the average of thepre-CE IPSPs. PST, postsynaptic transformation. The vertical arrowindicates the time of injection. Associating CE injection withpostsynaptic depolarization (0.4-0.6 nA during the off period with thecurrent intensities adjusted to elicit 4-8 spikes per s) transformsBAS-CA1 inhibitory PSP into an excitatory one (FIG. 10K) and produces afurther shift of the PSP-MP curve to the right (FIG. 10M). Associatingheat-inactivated CE with postsynaptic depolarization (0.4-0.8 nA at theoff period with current intensities adjusted to evoke 4-8 spikes per s)does not alter BAS-CA1 IPSP (FIG. 10L). Average responses of BAS-CA1 PSPafter associating either CE (CE+Ca²⁺) or heat-inactivated CE (Control)are shown in FIG. 10N. The transformed synaptic response is eliminatedby 1 μM bicuculline (FIG. 10O; 30 min).

Effects of CE on synaptic function were investigated on synaptic inputsfrom GABAergic BAS to CA1 pyramidal cells. Each GABAergic interneuronpowerfully inhibits some 1,000 pyramidal cells, providing widespreadcontrol over hippocampal networks (McMahon, L. L. & Kauer, J. A. (1997)Neuron 18, 295-305; Buhl, E. H., Halasy, K. & Somogyi, P. (1994) Nature(London) 368, 823-828; Cobb, S. R., Buhl, E. H., Halasy, K., Paulsen, O.& Somogyi, P. (1995) Nature (London) 378, 75-78). A singlepulsestimulation within the stratum pyramidale produced IPSPs in CA1pyramidal neurons at their resting membrane potential (FIG. 10A). TheIPSPs (−8.4±0.3 mV, n=8, P<0.05) were abolished (reduced by 95.1±3.2%,n=8, P<0.05, paired t test) by bicuculline (1 μM, 30 min), a GABA_(A)receptor antagonist (Sun 1996), indicating that the evoked IPSPs aremediated largely, if not exclusively, by activation of BAS-CA1 pathwayand are GABAergic. In some cases, a small but delayed inhibitorycomponent remained in the presence of bicuculline (not shown), possiblyrepresenting an incompleteblockade of the GABA_(A) receptor or GABA_(B)receptor-mediated component.

KYN (Sun et al. 1996; Collingridge, G. L. & Lester, A. J. (1989)Pharmacol. Rev. 40, 143-209), a competitive antagonist for bothN-methyl-Daspartate (NMDA) and non-NMDA ionotropic subtypes, receptorsfor the most dominant excitatory inputs to CA1 pyramidal cells, at 500μM (20 min; Collingridge et al. 1989) effectively eliminated SCH-CA1excitatory postsynaptic potentials (EPSPs) (by >90%) but did notincrease BAS-CA1 IPSPs (FIG. 10B). In the presence of KYN, the BAS-CA1IPSP (−8.1±0.4 mV, n=7, P<0.05) did not differ (P>0.05) from that ofpre-KYN (−8.0±0.3 mV, n=7, P<0.05), indicating the lack of a hidden,significant glutamatergic depolarizing component in the BAS-CA1 IPSPs.

The BAS-CA1 IPSPs were induced at different membrane potentials (FIGS.10C and D) and were found to reverse at a single membrane potential(−79.4±0.4 mV, n=59). No minor component was detected that exhibited adifferent reversal potential (FIGS. 10C and D). The relationship betweenBAS-CA1 PSPs and membrane potentials can be described with a straightline, not affected by KYN (FIG. 10E). The reversal potential in thepresence of KYN was −78.9 mV (±0.7 mV) and did not significantly differ(n=7, P>0.05) from pre-KYN values (−78.7±0.6 mV).

CE, applied postsynaptically into single pyramidal cells, produced alasting (>1 hr) reduction in BAS-CA1 IPSP (FIG. 10F and J; Table 1). Theeffect resulted from biological activity of CE, since heat-inactivatedCE was ineffective (FIG. 10G and J; Table 1). Membrane input resistancewas altered neither by CE (post-CE: 83.7±2.5 MΩ vs. pre-CE: 83.6±2.6 MΩ,n=10, P>0.05) nor by heat-inactivated CE (post-CE: 81.3±1.8 MΩ vs.pre-CE: 80.6±1.6 M, n=8, P>0.05). Microinjections (with the sameparameters used in the protocol) of CE conjugated with a greenfluorescent Alexa488 resulted in strong labeling of the cell body and aportion of the dendritic tree in the plane of focus (FIG. 10I1),indicating the efficacy of the CE microinjections. Similar intensity oflabeling of the cells was observed when heat-inactivated conjugated CEwas microinjected (FIG. 10I2). Effects of CE on K⁺ channels were evidentas a reduction in after-hyperpolarization (from −4.7±0.3 mV to −0.5±0.2mV, n=10, P<0.05) and a prolongation of the interval between briefintracellular depolarizing pulses (1 ms, 1-4 nA) sufficient to evokeaction potentials and the time required for the membrane potential torepolarize to its prestimulation level (from 39.5±1.9 ms to 53.4±2.3 ms,n=10, P<0.05).

CE-induced reduction of BAS-CA1 PSPs does not appear to result from asimple blockade of a receptor-channel complex. Rather, CE caused a shift(FIG. 10H) of the relationship between BAS-CA1 PSPs and membranepotential to the right and of the reversal potential to more positivepotentials (Table 1). The slope did not vary significantly on average.Heat-inactivated CE produced no such effect (Table 1).

TABLE 1 Effects of CE on BAS-CA1 PSPs of CA1 pyramidal cells % PSPsReversal potential, mV Treatment N Control Test P Control Test P CE 10−101.1 ± 42  −49.9 ± 47*  −79.0 ± 1.0 −70.1 ± 1.4* CE (I)  8 −102.3 ±3.5 −106.0 ± 3.6^(NS) <0.05 −77.6 ± 2.8  −78.0 ± 2.4^(NS) <0.05 CE +Ca²⁺ 10 −102.3 ± 2.4 +21.1 ± 4.5* −77.5 ± 0.9 −66.4 ± 1.8* CE (I) + Ca²⁺ 8 −102.5 ± 33   −99.2 ± 2.5^(NS) <0.05 −78.5 ± 1.6  −78.8 ± 1.7^(NS)<0.05 CE + Ca²⁺  7  −99.8 ± 2.3 +23.4 ± 3.7* >0.05† −78.9 ± 1.2 −67.2 ±2.0* >0.05† (BZA) Anti−CE  8 −100.4 ± 31  −121.9 ± 5.2* −79.5 ± 0.9−84.6 ± 2.2* Anti−CE (I)  7  −98.9 ± 2.9  −100.9 ± 3.1^(NS) <0.05 −79.2± 1.2  −79.6 ± 1.8^(NS) <0.05 (I) heat−inactivated form; BZA, bathbenzolamide. *, Significant difference (P < 0.05) as compared withpretreatments. ^(NS), no significant difference (P > 0.05) as comparedwith pretreatments. P indicates the significance of tests between thetwo groups; †, as compared with CE+ Ca²⁺ group. Control values wereobtained approximately 5 min before, while the test values were observedabout 30 min. after the application of the proteins.

When CE microinjection was coincident with postsynaptic depolarization(0.4-0.6 nA during the interval between injection episodes, to loadCa²⁺), the BAS-CA1 PSP was reversed to excitatory (FIGS. 10K and N andTable 1). This synaptic transformation lasted more than 1 hr (FIG. 10N)and did not occur suddenly, but rather as an extension of an initialgradual reduction in BAS-CA1 IPSPs (FIG. 10N). The transformed synapticresponse was eliminated by bath application of bicuculline (1 μM; 30min; FIG. 10O; by 95.6%±5.2%, n=6, P<0.05), indicating GABA_(A) receptormediation. The relationship between BAS-CA1 PSPs and membrane potentialshowed a further significant shift to the right with CE-depolarizationpairing (FIG. 10M). Co-application of postsynaptic depolarization withheat-inactivated CE, however, had no such effects (FIGS. 10L and N;Table 1).

FIGS. 11A-E demonstrate that anti-CE antibody enhances BAS-CA1 IPSPs andshows mechanisms of CE-induced transformation of GABAergic synapses.Anti-CE antibody injection into a recorded CA1 pyramidal cell enhancesBAS-CA1 IPSP (FIG. 11A, as compared with unmarked IPSP before injection)and elicits a shift of the PSP-MP curve to the left (FIG. 11C).Injection of heat-inactivated antibody is ineffective (FIG. 11B; twotraces overlapping). Average responses of BAS-CA1 PSP after injection ofeither anti-CE antibody (Anti-CE) or its heat-inactivated form (Control)are shown in FIG. 11D.

A schematic drawing (FIG. 11E) shows mechanisms of CE-mediatedtransformation of GABAergic synapses. Synapse-transforming signals (suchas associative activation of cholinergic and GABAergic inputs) turn on aCE/CE-like protein signal cascade. CE binds to the RyR and causes Ca²⁺release. The Ca²⁺/CE transforms the GABAergic synapses by shifting theGABA_(A) reversal potential from Cl⁻ reversal potential toward HCO₃ ⁻reversal potential, through altering anion selectivity of the Cl⁻channels, activity of CA, and/or formation of HCO₃ ⁻. Multiple arrowsindicate possible involvement of unidentified mediators. AA, arachidonicacid; DAG, diacylglycerol; ER, endoplasmic reticulum; PKC, proteinkinase C.

Microinjections of anti-CE antibody, which recognizes CE-like proteinsin rat and rabbit cerebellum and other brain regions, including thehippocampus, into CA1 pyramidal cells produced a period of enhancedBAS-CA1 IPSPs (FIGS. 11A and D and Table 1), whereas heat-inactivatedanti-CE antibody was ineffective (FIGS. 11B and D and Table 1). Thedifference, though small, was significant (Table 1). The anti-CEantibody-induced enhancement of BAS-CA1 IPSPs is not simply an increasein postsynaptic response for a given membrane potential. Therelationship between BAS-CA1 IPSPs and membrane potential was shifted tothe left (FIG. 11C). Thus, the antibody induced a significant change inthe reversal potential to more negative potentials (Table 1), whereasthe heat-inactivated antibody was ineffective (Table 1). The membraneinput resistance was not affected by microinjection of anti-CE antibody(post-antibody: 79.0±3.2 MΩ vs. pre-antibody: 79.2±2.8 MΩ) or itsheat-inactivated form post-inactive antibody: 80.7±4.1 MΩ vs.pre-inactive antibody: 80.6±3.0 MΩ). The IPSP enhancement by anti-CEantibody supports the idea that effects of CE on synaptic function arenot an artificial change of synaptic function, but involve effects onendogenous substrates within the CA1 cells.

According to the invention, reducing the GABA_(A) receptor-channel Cl⁻current and increasing the HCO₃ ⁻ current contributes to GABA-induceddepolarization (Staley, K. J., Soldo, B. L. & Proctor, W. R., Science269, 977-981 (1995); Grover, L. M., Lambert, N. A., Schwartzkroin, P. A.& Teyler, T. J., J. Neurophysiol. 69, 1541-1555 (1993)), and the lattershould be sensitive to CA inhibitors. HCO₃ ⁻ formation is a slow processbut is increased at least several thousand fold by CA (Dodgson, S. J.,Tashian, R. E., Gros, G. & Carter, N. D. (1991). The Carbonic Anhydrases(Plenum, N.Y.)), which is present within CA1 pyramidal cells (Paternack,M., Voipio, J. & Kaila, K., Acta Physiol. Scand. 148, 229-231 (1993)).The HCO₃ ⁻ reversal potential is about −12 mV (Staley et al. 1995) sothat an outward flux would result (FIG. 11E) and thus depolarizes themembrane at resting membrane potentials.

As seen in FIGS. 12A-K, ACET and non-bicarbonate buffer eliminateCE-induced transformation, and the transformation converts excitatoryinput filter into amplifier. ACET (1 μM) eliminates CE-induced synaptictransformation (FIG. 12A). The effect of CE on BAS-CA1 IPSPs is notobserved in Hepes buffer (FIG. 12B). In the presence of extracellularbenzolamide (10 μM), CE depolarization induces the synaptictransformation (FIG. 12C), which is not induced when BAPTA is co-applied(FIG. 12D; the charges carried by BAPTA are compensated by reducing theamount of acetate). Single-pulse stimulation (FIG. 12E) of BAS-CA1evokes an IPSP and of SCH at above-threshold intensities, actionpotentials (truncated; two traces: one stimulated at delay of 10 ms andthe other 30 ms, marked with arrows). The excitatory SCH (at the sameabove-threshold stimulation) input is filtered out by a costimulation ofBAS-CA1 (FIG. 12F; two overlapping traces). (FIG. 12G) Single-pulsestimulation of BAS-CA1 evokes an IPSP, and stimulation of SCH atbelow-threshold intensities evokes an EPSP. The excitatory SCH (at thesame below-threshold stimulation) input is below threshold as evoked bycostimulation (single pulse) of BAS-CA1 and SCH inputs (FIG. 12H) beforeCE application. CE (30 min after the application) transforms BAS-CA1IPSP and does not change much of the SCH-CA1 EPSP, evoked bysingle-pulse stimulation of BAS or SCH, respectively (FIG. 12I). Theexcitatory SCH (at the same below-threshold stimulation) input isamplified by the co-BAS stimulation after the CE-induced synaptictransformation and induces action potentials (truncated; FIG. 12J: twooverlapping traces). (FIG. 12K) Schematic diagram of transformedGABAergic synapse functioning as either excitatory filter (surround) oramplifier (center). Active BAS GABAergic inputs effectively filterexcitatory signals so that only very strong excitatory inputs mightevoke action potentials. The GABAergic synaptic transformation resultsin amplifying excitatory signals so that weaker inputs can pass throughthe neural circuits (through the cell in the middle). BAS, basketGABAergic interneurons (in black); Pyr, CA1 pyramidal cells.

In the presence of ACET (1 μM, 30 min), a CA inhibitor, CE caused noobvious alterations in the BAS-CA1 IPSPs (FIG. 12A; −99.2±3.5%, 30 minafter CE injection as compared with −100% control value, n=8, P>0.05).CA isoforms (such as cytoplasmic types I, II, III, and VII; cell-surfacemembrane type IV; mitochondrial type V; and secretory type VI; Landolfi,C., Marchetti, M., Ciocci, G. & Milanese, C. J., Pharmacol. Toxicol.Methods 38, 169-172 (1997); Linskog, S. Pharmacol. Ther., 74, 1-20(1997)) are zinc enzymes and show different sensitivity to ACETinhibition. Their activity can be regulated by hormones through cAMP inother tissues. Inhibition of probably the type II isoform (IC₅₀=0.09 μMfor ACET; Landolfi et al. 1997), in addition to a partial inhibition ofother less sensitive isoforms, appears effective in suppressing the CEeffect. Bath perfusion of the membrane-impermeant CA inhibitorbenzolamide (10 μM) was found to have no effects on the CE-inducedsynaptic transformation (FIG. 12C and Table 1), indicating that theinhibitory effects on CA were intracellular. At 10 μM (30 min), ACETitself was sufficient to transform the BAS-CA1 IPSPs, while abolishingeffects of CE on the synaptic response (n=8, not shown). Whennon-bicarbonate buffer was perfused externally, a condition to minimizebicarbonate effects, CE did not elicit any obvious changes in BAS-CA1IPSPs (FIG. 12B; −98.9±4.2%, 30 min after CE injection as compared with−100% control value, n=7, P>0.05). The cellular mechanism underlying theCE-induced GABAergic synaptic transformation thus involves an inductionof a depolarizing HCO₃ ⁻ flux through the GABA_(A) receptor-Cl⁻ channel(FIG. 11E). The effectiveness of CA inhibitors and of minimizingbicarbonate influence indicates that altered Cl⁻ accumulation throughchanged activity of K⁺—Cl⁻ transports is unlikely to be involved in thesynaptic transformation. CE/Ca²⁺ may induce changes in anion selectivityof the Cl⁻ channels, activity of CA, and/or formation of HCO₃ ⁻ (FIG.11E). Ion permeability of channels has been previously shown to bemodifiable by intracellular messengers or cations/anions (Williams, K.,Pahk, A. J., Kashiwagi, K., Masuko, T., Nguyen, N. D. & Igarashi, K.(1998) Mol. Pharmacol. 53, 933-941; Rychkov, G. Y., Pusch, M., Roberts,M. L., Jentsch, T. J. & Bretag, A. H. (1998) J. Gen. Physiol. 111,653-665). Ca²⁺- and ATP-activated HCO₃ ⁻/Cl⁻ conductance has beenobserved in nonneuronal cells (Ishikawa, T. (1996) J. Membr. Biol. 153,147-159). While these results strongly suggest a central role of HCO₃⁻/CA activity in the CE-induced synaptic transformation, CE does notappear to directly affect CA activity, as determined by measuring pHchanges in the reaction for CO₂ conversion to bicarbonate in thepresence of 4.4 nM CE, 20 μM Ca²⁺, and 6.6 nM CA (384±18 mmol/min·mg ofCA, n=4; as compared with 395±9 mmol/min·mg of CA in the presence of 4.4nM CE and 20 μM Ca²⁺, n=6; P>0.05). Nor was CA activity in the rat wholebrain homogenate affected by 4.4 nM CE and 10 μM Ca²⁺ (1.32±0.07mmol/min·mg of protein, n=2; as compared with 1.37±0.08 mmol/min·mg ofprotein in the presence of 4.4 nM CE only, n=2; P>0.05). It thus remainsto be determined what factors (coupled to CE and/or bound to Ca²⁺),which would be diluted to {fraction (1/10,000)} in the brain homogenate,might affect CA and thereby mediate the responses in untreated cells.Retrograde inhibition of GABA release (Alger, B. E., Pitler, T. A.,Wagner, J. J., Martin, L. A., Morishita, W., Kinov, S. A. & Lenz, R. A.(1996) J. Physiol. (London) 496, 197-209; Marty, A. & Llano, I. (1995)Curr. Opin. Neurobiol. 5, 335-341), as induced by depolarizing themembrane potential to +60 to +90 mV, might not be involved in theCE-induced inhibition of the GABAergic IPSP that follows negativecurrent pulses used to inject CE alone. Inhibition of GABA release wouldnot be expected to produce a reversed membrane response in polarity, norwould it be expected to be sensitive to CA inhibition or non-bicarbonatebuffering. The transformed response has also been observed in responseto externally applied GABA (Collin, C., Devane, W. A., Dahl, D., Lee, C.-J., Axelrod, J. & Alkon, D. L. (1995) Proc. Natl. Acad. Sci. USA 92,10167-10171) when paired with postsynaptic depolarization. ExogenousGABA (thus not under presynaptic control) delivered in the same quantitysubsequently produced depolarization rather than hyperpolarization.

CE binds to the RyR in neurons and induces intracellular Ca²⁺ release,as monitored with bis-fura-2 ratiometric imaging in rat CA1 pyramidalcells (Alkon et al. 1998). We, therefore, examined whether the RyR maymediate effects of CE on BAS-CA1 synapses. RR, a membrane-impermeantpolycationic molecule, inhibits the RyR with IC₅₀ in the nanomolar rangeand may alter its structure at micromolar concentrations. Itsspecificity is indicated by its lack of obvious effects on inositoltriphosphate receptor-mediated Ca²⁺ release and ensured by postsynapticapplication into singly recorded pyramidal cells (5 min before CEinjection). RR slightly increased the evoked BAS-CA1 IPSPs (by16.3%±4.0%, n=10, P<0.05), and it effectively blocked effects of CE onBAS-CA1 PSP (n=10). It neither reduced the GABAergic synaptic responsenor affected postsynaptic membrane properties (n=10). Such changes wouldotherwise be expected if a significant amount of RR permeated (frominside the cell) the membrane to block voltage-sensitive Ca²⁺ channelsand presynaptic transmitter release during the experimental period.Buffering intracellular Ca²⁺ with BAPTA mimicked RR in increasing theevoked BAS-CA1 IPSPs (by 17.4%±3.9%, n=6, P<0.05; FIG. 12D) and inblocking the effects of CE postsynaptic depolarization on the BAS-CA1PSPs (n=6). These results, together with the previously obtainedobservations that CE induces intracellular Ca²⁺ waves in hippocampal CA1pyramidal neurons and release ⁴⁵Ca²⁺ from microsomes (Alkon et al.1998), indicate that CE regulates intracellular Ca²⁺ levels. Theeffectiveness of RR and BAPTA, however, does not rule out thepossibility that Ca²⁺ might function as a cofactor for CE and/or othermediators to induce changes in anion selectivity of the Cl⁻ channels andactivity of CA.

The BAS interneurons in CA1 are part of hippocampal networks thatcontrol the main excitatory input pathway and thus play a critical rolein determination of information processing in CA1 pyramidal cells andmemory storage, including transmission of the theta rhythm from septunto the hippocampus (Toth, K., Freund, T. F. & Miles, R. (1997) J.Physiol. (London) 500, 463-474). In a separate study, we observed thatGABAergic synaptic transformation can be induced through associativeactivation of the cholinergic-GABAergic inputs into the CA1 pyramidalcells (unpublished observations). Thus, CE-induced transformation ofGABAergic synapses might help determine the synaptic effect of thecholinergic system during attention to training-induced stimulusassociation (Fisahn, A., Pike, F. G., Buhl, E. H. & Paulsen, O. (1998)Nature (London) 394, 186-189).

GABAergic interneurons receive excitatory inputs from SCH/commissuralafferents in a feed-forward manner (Buhl et al. 1994; Cobb et al. 1995;Paulsen, O. & Moser, E. I. (1998) Trends Neurosci. 21, 273-278) andpreferentially make synapses on cell bodies, proximal dendrites, andaxon initial segments of CA1 pyramidal cells (Cobb et al. 1995; Halasy,K., Buhl, E. H., Lorinczi, Z., Tamas, G. & Somogyi, P. (1996)Hippocampus 6, 306-329). One BAS cell is estimated to have over 10,000boutons innervating some 1,000 pyramidal cells (Halasy et al. 1996),forming 10-12 synapses on each pyramidal cell (Buhl et al. 1994; Cobb etal. 1995). The perisomatic termination of BAS cells is suited forsynchronization of pyramidal cells (Cobb et al. 1995). Modifiability ofinhibitory circuits may thus be less specific but more efficient incontrolling a specific population of pyramidal cells (Cobb et al. 1995).Furthermore, the coincidence of GABAergic and the more specificglutamatergic inputs could confer great specificity in a center-surroundmanner (FIG. 12K). The transformed synaptic input from the BAS cellscould provide a mechanism to selectively activate a subset of pyramidalneurons (those transformed and thus in the “center” of attention) andblock others (those not transformed and thus in the “surround”).Activation of BAS produces fast IPSPs and reduces excitability (FIG. 12Eand F) and probability of action-potential generation (Andreasen, M. &Lambert, J. D. (1998) J. Physiol. (London) 507, 441-462) of CA1pyramidal cells. SCH stimulation at intensities above (30%) thresholdelicits action potentials (100% of 10 trials; FIG. 12E). BAS stimulationproduced an effective signal-filtering period of 50-100 ms (up to 200 msin some cases), during which no action potential (0% of 10 trials) wasevoked by SCH stimulation at the same intensities (FIG. 12F; n=9,P<0.05). Action potentials were reliably elicited (FIG. 12J; n=9) bysingle-pulse costimulation of SCH (at below threshold intensities) andBAS after CE-induced transformation (FIG. 121 as compare with FIG. 12G).Before the CE application, the same intensities of costimulation did notevoke action potentials (FIG. 12H; n=9). Weak signals are amplified inthe transformed cells, whereas only very strong excitatory signals cansuccessfully pass through the network under BAS inhibition. Thus,opposite GABAergic effects in subsets of neurons could act as eitherfilter or amplifier, increasing the signal-to-noise ratio of relevantinformation, that is in the focused center of attention (FIG. 12K).

Discussion

Transformation of GABAergic inhibitory into excitatory synapticpotentials has been observed experimentally by several groups, withtransformed response lasting either for a short term (seconds) tominutes. (Staley et al. 1995; Wong, R. K. S. & Watkins, D. J. (1982) J.Neurophysiol. 48, 938-951; Kalia, K., Larnsa, K., Smimov, S., Taira, T.& Voipio, J. (1997) J. Neurosci. 17, 7662-7672; Taira, T., Lamsa, K. &Kaila, K. (1997) J. Neurophysiol. 77, 2213-2218) or long period (≧1 hr;20; Alkon, D. L., Sanchez-Andres, J. -V., Ito, E., Oka, K., Yoshioka, T.& Collin, C. (1992) Proc. Natl. Acad. Sci. USA 89, 11862-11866; Alkon,D. L., Lederhendler, I. & Soukimas, J. J. (1992) Science 215, 693-695).While fascinating and important because the transformation results in anovel synaptic response, its role in memory, and the intracellularsignaling cascades that lead to the synaptic transformation werepreviously unknown. The present study provides evidence that lastingchanges in synaptic polarity can be orchestrated by CE, an associativememory-related signal protein (Alkon et al. 1998; Nelson et al. 1990).CE switches GABAergic synaptic function from excitation filter toamplifier and may help control hippocampal networks andhippocampus-dependent memory processing.

The embodiments illustrated and discussed in this specification areintended only to teach those skilled in the art the best way known tothe inventors to make and use the invention. Nothing in thisspecification should be considered as limiting the scope of the presentinvention. The above-described embodiments of the invention may bemodified or varied, and elements added or omitted, without departingfrom the invention, as appreciated by those skilled in the art in lightof the above teachings. It is therefore to be understood that, withinthe scope of the claims and their equivalents, the invention may bepracticed otherwise than as specifically described.

What is claimed is:
 1. A method of suppressing or enhancing attentivecognition in a mammal in need thereof comprising administering to themammal a compound that modulates carbonic anhydrase activity in thebrain in an amount sufficient to suppress or enhance attentivecognition.
 2. The method of claim 1, wherein the compound inhibitscarbonic anhydrase activity in the brain and suppresses attentivecognition.
 3. The method of claim 2, wherein the compound is selectedfrom the group consisting of acetazolamide, benzolamide, and analogsthereof.
 4. The method of claim 2, wherein the compound preventsestablishment of a theta rhythm.
 5. The method of claim 2, wherein thecompound prevents formation of associative memory.
 6. The method ofclaim 2 wherein the suppression of attentive cognition is specific. 7.The method of claim 1 wherein the compound modulates intraneuronalcarbonic anhydrase activity.
 8. The method of claim 1, wherein thecompound modulates extraneuronal carbonic anhydrase activity.
 9. Themethod of claim 1, wherein the compound alters neuronal HCO₃ ⁻conductance.
 10. The method of claim 1, wherein the compound modulates aneuronal HCO₃ ⁻ current relative to neuronal Cl⁻ and/or K⁺ currents. 11.The method of claim 1, wherein the compound strengthens theta rhythm inthe mammal.
 12. The method of claim 1, wherein the compound promotessynaptic transformation in the mammal.
 13. The method of claim 1,wherein the compound increases or decreases bicarbonate-mediatedGABAergic depolarization.
 14. A method of improving attentive cognitionin a patient in need thereof comprising administering to the patient acompound that stimulates carbonic anhydrase activity in the brain in anamount sufficient to improve attentive cognition.
 15. The method ofclaim 14, wherein the patient has no neurodegenerative disease ordisorder.
 16. The method of claim 14, wherein the patient suffers from aneurodegenerative disease or disorder.
 17. The method of claim 14,wherein the compound stimulates intraneuronal carbonic anhydraseactivity.
 18. A method for treating a neurological disease or disorderin a mammal comprising administering to the mammal a stimulator of braincarbonic anhydrase activity in an amount effective to improve attentivecognition.
 19. The method of claim 18, wherein attention and/or memoryacquisition is enhanced.
 20. The method of claim 18, wherein theneurological disease or disorder is selected from dementia, stroke,hypoxia, and ischemia.
 21. A method of stimulating synaptictransformation of inhibitory postsynaptic potentials into excitatorypostsynaptic potentials in GABAergic synapses in a mammalian brain,comprising administering to the brain an activator of intraneuronalcarbonic anhydrase activity, thereby stimulating the synaptictransformation in the synapses.
 22. The method of claim 21, wherein thesynapses are in pyramidal cells in the hippocampal region.
 23. A methodof blocking synaptic transformation of inhibitory postsynapticpotentials into excitatory postsynaptic potentials in GABAergic synapsesin a mammalian brain, comprising determining a need for blocking, andadministering to the brain an inhibitor of intraneuronal carbonicanhydrase activity, thereby blocking the synaptic transformation in thesynapses.
 24. The method of claim 23, wherein the inhibitor neutralizesthe excitatory effects of calexcitin.
 25. The method of claim 23,wherein the inhibitor is acetazolamide or an analog of acetazolamide.26. The method of claim 23, wherein the synapses are in pyramidal cellsin the hippocampal region.